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Kinetic Analysis and Intermediate Structure Determination From High-Speed Time-Resolved Crystallography

Authors:
  • Acculation, Inc.

Abstract and Figures

Despite the very recent availability of High Speed Time-Resolved Crystal­lography (HSTC) data sets, the promise of HSTC remains largely unfulfilled due to the difficulty in extracting structural information on intermediates from time-resolved electron density maps. These maps are only composites of the electron density maps of the populated chemical species present at the experimental time points. Although various schemes of extracting homogeneous structures from HSTC data sets have been proposed, to date no structures have been determined. In this paper we present novel methods for estimating these parameters directly from an HSTC data set. We apply our methods to successfully extract structural information on intermediates from an HSTC data set acquired during decay from the saturated photostationary state of the Photoactive Yellow Protein (PYP). We present the refined structure (pseudo-2.0 Angstrom resolution by estimates of completeness) for a previously unseen intermediate of the PYP photocycle decay.
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TABLE OF CONTENTS
 
vi
      
viii
  
ix
   
x
 
xi





  

   
           
 


          



                 
6
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    
        

                   

                 
       
      

           

       

 
   

          


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           

        
    
iv
                      

  

                     

       

           
 
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                           ,1s
  
  

               
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  
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 
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82
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  
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
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
             
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 
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 
                

  
         

    

  

   


 

  
x



   x



               



   

   

 

    

     










 
                      
vi
Figme


                             


  
                         

    


  


     
                        


            



 

   


   
 
                          



                            

    
                            

   
   
            


 
             
vii
LIST OF TABLES


 
  




   


      







  

37

 

   
                        


 

   
                     



                       
  


                        
viii
  





ix


  
  
     
    
    
    
       
 

 
       

1 

A   

x

 
        
           
           
         
          
          
     to       
 
xi
 

     (1)     
 

  
 
       
      
 
       
   
 
        
    

    
 
   
     
  
          
       


 
          
     
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   

     
 

           
         
  
       
      

        
      
   
        


   



       
        
       
  
      
        
 
 

          
    
      

3

 
  
           
  
         
 

    
   
      et al.    

  
  

     
  
r
    
 
 
    
  
  
  


    

  + 

   
        




  
 
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     
0

  

 
 
     
       
 
 t) 
 
    

 
    
     




           
 


i,j =













± 


   
        
      
 



+ 

 














 
 +
1

 




   






 

 « 
+ 

 
 »
+ 











 
  
   
8
           


 

  

C; 

 
      



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


       n     
     t,  
          
 
    
          
         
       
    
 
i
g

 
       
            
     
           
       
 

    






       
           

      


  


    
   
 
     
 
      
  

  
 

 

   
 

  







(n
1
   

Exponents
        
   
          
 
 

 

       
      
 


12
 T          
         n    
        
+ + 
n 

       
 n )n  
 n + 
    

     
  
 

  
 n( n  

   
 

  
         
          
         
+          
+ 
  
   
13


   
 

 

  
  






A
,
n
-
1

A
1
,
n




































Am-l,n-1













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
Am.,n-1

-
m
,
n






 
L

 
L
 
dt 






',J
 

j 


        

 
+
L





    

B).
 


J






  



L
[c + 

L 


J
j
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  
 

16
         
    p. 
         

      
          
          
       
      
   
          
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         
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
        
        

 
           
    
17

      
        
 
   
rem:
     
        


       
      
              
          
       

         
          

     
         
     
         
        
     
   
01




 

  



 
    



[A]= { + 
1







(2.13)


 
   





 




19




 
01 


 






IF(k, t)1
2














j

i
,
j





j



id

i



IF(k,t)l
2
= (
~!c,Fc,(k))
· (
~/c;Fc;(k)).
IF(k, t)l
~
LfcilFcJk)I,
i
20
 







           
 t.
  
   
  
 


 


t.
        
  

  
    
 

  
 
    

     
        

      


         

 
 
     



    
    

X 
           
          
         
      
        
      
T
    
        
 \,            
C E. 
 
6

     
3
    


  

  
            
          
         

           
        
C E, 
 
C E  

  
     
     
10
  
   

  
 
 
 
           

 
      
 
 
 

       
   
      
   
     
   vV.       
          
  


      
 
3

     
        

       
 
ncomp·    
3
)  
     
 


  
        




3


 

 



 











        
        
        
      
 

     
    
        
      
     
    
 



         
   
     
   

   
        
 

      
       
  
     
1
 

  
4
2
          

3
  
2
   
 
  
          
  
r
     
         
(n) 
 
 
26

  

     
            
            
     
    
          
      
  
   
  
  


          
         
    
 

  
       A
i
(k)  
     
    
        

         
   
 
   
 



   
   

    
 
 
    
      
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 

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      
 
 
  
 
     
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          
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        
 
  

     

 

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 
    
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 fc.        
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n  n  c, 
   
 

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   
         
         


  
       



    
    
      J
0
,( t)  
 I          

1
2


        
   Z,       
          

2

~.fcilFci(k)I
~
L~(k)exp[-kit].
i i
29
 
    

L

L 

    L 


  

n


L



1
2
i
,
J
        
        
   
       

        

 to 
  
         
           
  
  


     
   
- LLIFon(k)l
2
30
 
 
 
       
     

          
       
     
          
   
    



     t        
     
  
    
           

         
    
        
       

        
     
    
31

      

  

         
        
     

       

 

       
 



    
      


          
   t         
     
  
  
32


  


 

.




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
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


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
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

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


        

        
      
 
I:
I
 
    

 
    
 
  
      
 

 
    
     
   
   

 
         
          (k =   
3

1
)
  (k   
   
  
      to      
 
    
         


         
     A
   
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

native






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




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c
c
c
c




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

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
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

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
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


2
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

9
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


2
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
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


 
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
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 
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 
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
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 
 
  


     
        
          
 

 
      
           
      
            

          
     

            
 
            
          
      

     

 

 

 

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 

   
  

        

   
  
        Laue ew 



 
  
       
      
         
            
        
  
 
 

 
 

 
     
           
       
         
        
      


  
            
          
         
 
   
 
     
            
       
 
  
   5 
       
        
           
        

   


            
         

 
  

   
         
      
         
         
     
  
      
             
      
       et al.    
         
         

 
    

         
          
      



 
    
 
  
       

   
 
  
      
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
    
 


       
 
     
 
   
 


  
          

  

        
     


        

     et al.
     
          
     
        
        

        
47
   
   
          
     
            
       
     
         
        
       

         
           
         
  
          
     p,   
     
   
1
 
   

 

        

  
      
         

 
 

         

 

   et al.     
     
       
            
 

   
  
  
         
         
          

    
 
   
            

A B,
(3.1)


    
   
-
1
9
 






 

 
    

0
.1
o.,
0 . 2
50
 






   

       
       

 
0 . 1
51
n

c
1

1
X 




c
1


    IF c
1
 
2 
     
      
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     
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 
  
 
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
         
          

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          
 


    
       
      
    
       

   
 
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


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      
     
          
           
         
     
        

 
   
f 
 
   
         
 

 
     
  
 

53
    
     

   
1
   
        
   
  


 

 
 
      
         
 
       

  

   
      
A 
        
 
  
  
 
   
  

54
   


  
       
         
         
 
           
  

          
       
          
 


       
     
          
         
      


   
   (1)  

       

 
      
        
   
         
  
 

  
           
   

56
       

 
      
 

          

     
          
  
          
         
 
   

         
 

 
       
       
         

         O(n) 
         
  
         
         

 
 
    
         
 

     
 
         
   
  

 
 
A 
 
58

   
   



 
   



  


  
  

  

Fie 7        
        
    
   
           
              
    
              
  
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) 7 8 0 chisq:547.873
10
UJO
1000
10IX!O
60
1: ( #751 ) 4
41
chisq:427.173 2: ( #1220) 6 9 8 chisq:458.677 3: ( #979) 5 6 4 chisq:541.319
10 1ix, 1000 10000
111!'11
{
1!".1
)
5: (
"15
) 0 2 5 chisq:551.744
10
100
1000
10000
ON
(~ )
10
1
00
1000
1
0000
10
100
1000
t!JOO;I
""4
(~ )
tiff:
(~ }
6: (
"1169
) 6 5 7 chisq:749.543 7: (
"403
) 2 7 6 chisq:842.262
,o
100
,coo
10000
10
100
,coo
10000
8: ( #2348)
12
21
chisq:1345.18 9: (
"1565
) 8 3 6 chisq:1535.18 .
10
: ( "371) 2 5 1 chisq:1642.9
10
100
1000
10000
10
100
1000
tOOO!J
TO
t
OO
HIOC
I 1
0000
-----
Figure
9.
Individual reflections
with
worst-qualtity fit. These
ten
reflections
have
the
worst-quality fits
to
the
global exponential decay
parameters.
They
were
determined
by
the
same
process
as
in
the
previous two figures. Outliers
can
adversely affect
the
global exponential fitting process by disproportion-
ately affecting
the
aggregate figure-of-merit process used
to
fit
the
global
exponential decay parameters.
_,,,
...
,,.
--
r•o
-
....
..,
-·
I r.>77
)1
12
5
ChiO<F
..
7001
12. i 1588) J 5 8 chioc,pe 53831
• !"'•1
1 (
il1"2)
1 1
11
chil<l"J 34708
( 11321121•chio<F:184128
I
I]
'
OI
1
000
OQQO
t'l"'
l l,..t)
61
2 ( 11•J> 1 1
12
Ohi9"'5.2812
, : (
111111
) 1 5 12 chit
Cl"10
791
7 I
'412)
2
81
ohio<F1J
a.407
10
1
00
,ooo
to«xi
....
~,
.....
. ,
....,
t
l'III
l\'IOO
•l'IOOO
1 (
"1
'
Figure 10. Individual reflection fits randomly selected. Depicted here
are
ten
individual reflections selected
at
random
from those fitted
by
the
non-linear
process
also depicted
in
the
preceeding
three
figures.
62

 20





 x
2
 
       
  
 

chi-squared
0
500 1000
1500
'··
·.
63


10
20
!Fl
      x
2
 
  

x
2

 
 
8
l'I
...
.
. · .
..
.
·._
::
..
,
K
"
'

'


'
k,
=
7500
   
       
 
   
   

 

      
1
.   
    

   
 
    
65

G
         


G
          
 





 

 
 
    

G
  

          

  
Pl
67
P2
G
      

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 
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 
 
     
        
         
     
       

     
        
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 
 
    
      
  
     
      
        
  

70
        
          

   
  
       
         
        
   
   
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71
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   
 
     
             


74
 

)
75
/
         

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76
 
 
77
         
        
          
          
        
          
  
78
  
        
 
79
     
  
 
    
     
 
)
80

  
       
 
)
I ,

      
   
            
   


  
 

  
    
         
    
     
     
          

     
      
 
  
      
   
   
   
        
 


     
   

    R     
   
   
 
  


          
     
 

         
  

  

  
  
        
        
   
 
  
        J.


    

     
 
            
       
    
 
            
 
   

        

 
  
  

      
 
            
   


   

   
     
   
    
   
           
 
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The reduction of X-ray diffraction data obtained by the Laue method to accurate integrated intensities is more complicated and much less familiar than the reduction of monochromatic data. Problems of data accuracy and completeness have hindered the wide use of the Laue technique in macromolecular crystallography. Its unique advantage, data-collection speed, has been exploited only in situations such as fast time-resolved crystallography, to which monochromatic techniques are not as well suited. This paper reviews the major problems in data reduction in the Laue technique and provides a unified solution to the problems in integration of both streaky and spatially overlapping spots and data scaling. This solution has been incorporated into a new Laue diffraction data-reduction software package, LaueView. Laue data sets from crystals of lysozyme and alpha-haemolysin have been processed to test this solution, and demonstrate that Laue data sets can be reduced to yield structure amplitudes of at the very least the same quality as the best monochromatic data sets in terms of both accuracy and completeness.
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The first essential step in protein photoreception is the capture and storage of energy from a photon. We have recently identified and isolated, from the purple photoautotrophic bacterium, Ectothiorhodospira halophila, a 13,000-dalton photoactive yellow protein (PYP) that has a photocycle with kinetics similar to sensory rhodopsin and a very high quantum efficiency. To study the structural chemistry of protein photoreception, we determined, refined, and analyzed the crystallographic structure of PYP at 2.4 A resolution and report here that it is composed of two perpendicular antiparallel beta-sheets that enclose the chromophore. Each of the 10 beta-strands of PYP is connected directly to its nearest neighbor with +1 topology. Globally, an asymmetric distribution of side chains places aromatic and acidic side chains in an ellipsoidal band around the chromophore with a cluster of basic side chains on one side. Locally, the electron density maps place an internal lysine and the chromophore in an apparent Schiff base linkage stabilized by a buried glutamate and a tyrosine side chain. To our knowledge, the atomic resolution structure of a protein with a reversible photoisomerization has not been reported previously. Furthermore, PYP may also represent a class of proteins that bind conjugated molecules and interact with a secondary receptor system.
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A photoactive yellow protein purified from the phototrophic bacterium Ectothiorhodospira halophila, has been crystallized by vapor diffusion from ammonium sulfate solution. The hexagonal crystals are in space group P6(3) with unit cell dimensions a = b = 66.89, c = 40.68 A and appear to have one 15,000-dalton protein in the asymmetric unit. Photoactive yellow protein contains a chromophore with retinal-like properties; its color can be reversibly bleached, by visible light, with kinetics similar to those of sensory rhodopsin. The crystals can also be bleached by an intense visible light source without cracking, but are not bleached by x-rays. This suggests that structures can be obtained for both bleached and colored conformations of the protein-bound chromophore. The crystals diffract strongly to at least 1.3 A resolution, are resistant to radiation damage, and are suitable for a high resolution structure determination. The covalently bound chromophore and photobleaching characteristics of the protein offer unique opportunities to study protein conformational change and refolding as well as to understand the mechanisms of light-induced conformational change at atomic resolution.
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The complete amino acid sequence of the 125‐residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4Åresolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86 , 6533‐6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light‐induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore‐binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+ 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.
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All chemical and biochemical reactions proceeed via very rapid changes in electronic and atomic structure. These changes may be visualised by ultrafast time-resolved crystallography, on time scales from a few hundred picoseconds to seconds or longer. The experiments exploit the pulsed nature of synchrotron sources, and their intensity and polychromatic nature. Applications to light-sensitive biological systems will be presented.
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