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Amphiphiles modify the properties of detergent solutions used in crystallization of membrane proteins
Abstract
The effect of the amphiphile heptanetriol on the properties of solutions containing several detergents commonly used for crystallization of membrane proteins was characterized. The critical micelle concentration was found to be relatively unchanged by the presence of the amphiphile. In contrast, the addition of heptanetriol to solutions containing both detergent and polyethylene glycol exhibited significant shifts in the clouding behavior, with the largest shifts being for lauryl dimethylamine oxide. These results suggest that conditions favorable for crystallization of integral membrane proteins can be inferred from the properties of the detergents and amphiphiles.
... Zwitterionic CNSL-based surfactants Three commercial surfactants (Dodecyldimethyl Betaine [150], Cocamidopropyl Betaine [151] and Lauryldimethylamine N-oxide [152]) were added in this table for comparison purposes. ...
Surfactants are crystallizing a certain focus for consumer interest, and their market is still expected to grow by 4 to 5% each year. Most of the time these surfactants are of petroleum origin and are not often biodegradable. Cashew Nut Shell Liquid (CNSL) is a promising non-edible renewable resource, directly extracted from the shell of the cashew nut. The interesting structure of CNSL and its components (cardanol, anacardic acid and cardol) lead to the synthesis of biobased surfactants. Indeed, non-ionic, anionic, cationic and zwitterionic surfactants based on CNSL have been reported in the literature. Even now, CNSL is absent or barely mentioned in specialized review or chapters talking about synthetic biobased surfactants. Thus, this review focuses on CNSL as a building block for the synthesis of surfactants. In the first part, it describes and criticizes the synthesis of molecules and in the second part, it compares the efficiency and the properties (CMC, surface tension, kraft temperature, biodegradability) of the obtained products with each other and with commercial ones.
... It is known that a subtle change of chemical structure of detergents used affects significantly the crystallization behavior of PDCs (Thiyagarajan and Tiede, 1994; Marone et al., 1999). Moreover, additives with small molecular weight (such as heptane triol) strongly affect the crystallization in such a way that they change the structure of PDCs themselves and interaction between PDCs (Timmins et al., 1991; Thiyagarajan and Tiede, 1994; Marone et al., 1999; Rosenow et al., 2001). In this study, however, we approximate PDC particles as simple spheres, ignoring their complex structure to focus on the effect of PEG on the interparticle interaction. ...
Based on the importance of crystallizing membrane proteins in a rational way, cytochrome bc(1) complex (BC1) was crystallized using polyethylene glycol (PEG) as a sole crystallization agent. Interaction between protein-detergent complexes of BC1 was estimated by dynamic light scattering, and was compared with the numerical calculation using the Derjaguin-Landau-Verwey-Overbeek potential plus a depletion potential, without considering specific surface properties of the protein-detergent complexes. The experiments and calculation were found to be consistent and we obtained a relation between PEG molecular weight M and the range of depletion zone delta as delta approximately M(0.48+/-0.02). The stability of liquid phase of BC1 solutions was controlled by a ratio of (the range of depletion zone)/(the radius of a BC1 particle), which was consistent with recent theoretical predictions. The crystallization was most successful under a condition where the stability of the liquid phase changed from stable to unstable. The PEG molecular weight that fulfilled this condition coincided with the one used empirically to crystallize BC1 in the past by a number of groups. These results are compared to the fact that membrane proteins were often successfully crystallized close to the detergent cloud point.
A specific mechanism for tethering micelles composed of non-ionic detergents is presented. The mechanism does not require any precipitant, high ionic strength or temperature alterations. Rather, it relies on complexes between hydrophobic chelators embedded within the micelle and appropriate metal cations in the aqueous phase, serving as mediators. The approach was applied to: (i) four non-ionic detergents (tetraethylene glycol monooctyl ether (C8E4), n-dodecyl-β-D-maltoside (DDM), octyl β-D-1-thioglucopyranoside (OTG), and n-octyl-β-D-glucopyranoside (OG)), (ii) two hydrophobic chelators (bathophenanthroline and N-(1,10-phenanthrolin-5-yl)decanamide, Phen-C10) and (iii) five transition metals (Fe2+, Ni2+, Zn2+, Cd2+, and Mn2+). The mandatory requirement for a hydrophobic chelator and transition metals, capable of binding two (or more) chelators simultaneously, was demonstrated. The potential generality of the mechanism presented derives from the observation that different combinations of [detergent:chelator:metal] are able to induce specific micellar clustering. The greater solubilization capacity of tethered-micelles in comparison with untethered micelles was demonstrated when the water insoluble aromatic molecule fluorenone (8 mM = 1.44 mg mL−1) and two highly lipophilic antibiotics: chloramphenicol (5 mM = 1.62 mg mL−1) and tetracycline (1.5 mM = 0.66 mg mL−1) were solubilized – only when the micelles were tethered.
With the advent of the recent determination of high-resolution crystal structures of bovine rhodopsin and human beta2 adrenergic receptor (beta2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and beta2AR. Structure determination of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biology becomes routine: over-expression, solubilization, and purification of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purification efforts, we review published conditions for solubilization and purification using detergents and additives. A discussion of sample preparation of GPCRs in detergent phase, bicelles, nanodiscs, or low-density lipoproteins is presented in the context of potential structural biology applications. In addition, a review of the solubilization and purification of successfully crystallized bovine rhodopsin and beta2AR highlights tools that can be used for other GPCRs.
The role of contact interactions in the crystallization of membrane proteins was assessed by mutation of amino-acid residues on the surface of the reaction center from Rhodobacter sphaeroides. Five single-site mutants were constructed, with changes in contact regions found in the trigonal and tetragonal forms but not the orthorhombic form. Crystallization trials for the tetragonal form yielded either no crystals or crystals with an altered morphology, whereas crystals grew in the other two forms, indicating that these interactions are essential for the stability of the tetragonal crystals. Changes in the structures determined by X-ray diffraction of trigonal crystals for each mutant were related to the quality of the diffraction. Significant differences in the resolution limit of the crystals were associated with the loss of specific interactions between neighboring proteins. The results suggest that the contact regions are crucial for obtaining highly ordered crystals of membrane proteins.
The relationship between the effect of detergents and amphiphiles on protein solubility and their use in crystallization solutions was examined for the reaction center from Rhodobacter sphaeroides. Measurement by a centrifugation assay of the solubility of the reaction center as a function of ionic strength revealed dramatic differences in the intrinsic solubility at zero ionic strength in the presence of various detergents and amphiphiles. High protein-solubility values were found for beta-octyl glucoside and for lauryldimethylamine-N-oxide with heptanetriol. The solubility differences are interpreted in terms of fundamental properties such as the polarity of the detergent molecules. Conditions that resulted in high protein solubility correspond to conditions that have been shown to be successful for crystallization of the reaction center. These results suggest that crystallization is favored for detergents and amphiphiles that optimize the solubility of integral membrane proteins.
Membrane proteins represent roughly one-third of the proteins encoded in the genome, yet fewer than 1% of the proteins are of known structure. High-throughput crystallography offers the hope of correcting this imbalance. In order for large-scale membrane protein structural biology to realize its full promise, however, significant technical challenges must be overcome, the two most substantial being facile protein overexpression and reliable methods for crystal growth.
The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol the first sterol precursor of cholesterol in mammals. Therefore, it is an important target for the development of new hypocholesterolemic drugs. Here, we report the overexpression and purification of functional human OSC (hOSC) in Pichia pastoris. The obtained IC(50) for the reference inhibitor Ro 48-8071 is nearly identical for the recombinant hOSC compared to OSC from human liver microsomes. The correlation of analytical ultracentrifugation data and activity measurements showed the highest enzymatic activity for the monomeric hOSC indicating that this would be the natural form. Furthermore, these data helped us to identify the detergent for a successful crystallization of the protein. The availability of this active recombinant human membrane protein is a very important step on the way to a more detailed functional and structural characterization of OSCs.
Although the examination of the protein data bank reveals an important backlog in the number of three-dimensional structures of membrane proteins, several recent successes are serving as preludes to what will become a very prosperous decade in this field. Systematic investigations of various factors affecting the stability of membrane proteins, as well as their potential to crystallize three dimensionally, have paved the way for such achievements. The importance of the role of detergents both at the level of purification and crystallization is now well established. In addition, the recognition of the protein-detergent complex as the entity to crystallize, as well as the understanding of its physical-chemical properties and discovery of factors affecting these, have permitted the design of better crystallization strategies. As a consequence of the various efforts in the field, new crystallization methods for membrane proteins are being implemented. These have already been successful and are expected to contribute significantly to the future successes. This chapter will review some basic principles in membrane protein crystallization and give an overview of the current state of the art in the field. Some practical guidelines to help the novice approach the problem of membrane protein crystallization from the initial step of protein purification to crystallogenesis will also be given.
A generic protocol that utilizes a dual hexahistidine-maltose-binding protein (His6-MBP) affinity tag has been developed for the production of recombinant proteins in Escherichia coli. The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification. The fusion protein (His6-MBP-passenger) is purified by immobilized metal affinity chromatography (IMAC) on nickel-nitrilotriacetic acid (Ni-NTA) resin and then cleaved in vitro with His6-tobacco etch virus protease to separate the His6-MBP from the passenger protein. In the final step, the unwanted byproducts of the digest are absorbed by a second round of IMAC, leaving nothing but the pure passenger protein in the flow-through fraction. Endogenous proteins that bind to the Ni-NTA resin during the first IMAC step also do so during the second round of IMAC. Hence, the application of two successive IMAC steps, rather than just one, is the key to obtaining crystallization-grade protein with a single affinity technique.
Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).
The process of crystallizing membrane proteins is hampered by several factors. Isolating significant quantities of membrane proteins is difficult because they are unstable and their biochemistry is not well understood. Even when the membrane proteins have been solubilized, several variables must be taken into account in order to achieve crystallization. The most important of these are the choice of detergent, and amphiphillic additives, as well as the type of lipid. Despite these difficulties, progress in being made, as the recent resolution of the plant light-harvesting complex structure has shown.
The three-dimensional crystals of membrane proteins can be divided into two classes: (1) type I crystals, which consist of layers of molecules in which the hydrophobic contacts between the lipid-embedded portions of a protein mediate the intralayer interactions and the hydrophilic contacts between the domains exposed to aqueous solvent define the interlayer interactions and (2) type II crystals, which are lattice contacts that primarily involve the polar domains of the protein; the hydrophobic regions are solvated by detergent micelles and do not participate in direct protein–protein interactions. Most membrane-protein crystals that diffract X-rays to high resolution belong to the type II category. The chapter emphasizes problems specific to the crystallization of membrane proteins and discusses crystallization studies of α-hemolysin (α-HL) from Staphylococcus aureus as a model protein for studying membrane-protein crystallization.
The crystallization of membrane proteins is described. The topics covered
include the principles of membrane-protein crystallization, general
properties of detergents relevant to membrane-protein crystallization, the
`small amphiphile concept', membrane-protein crystallization with the help
of antibody Fv fragments and membrane-protein crystallization using cubic
bicontinuous lipidic phases. General recommendations on crystallizing
membrane proteins are provided, and membrane proteins with known structures
and potentially useful detergents are tabulated. This chapter is also
available as HTML from the International Tables Online site hosted by the
IUCr.
After a long period of fruitless attempts, three-dimensional crystal of membrane proteins are now available. The best of the crystals are suited for high-resolution structure determination by X-ray crystallography. The use of small detergents and small amphiphilic molecules during crystallization proved to be of great value.
The presence of small amphiphiles has been found to be necessary in the crystallization of several membrane-protein/surfactant complexes. It has been suggested that the role of the small amphiphile may be to reduce the size of the surfactant belt around the protein, making the formation of crystals easier. Thus far it was not known if this would involve changes in micellar size in general or whether the small amphiphile would merely replace LDAO during crystal growth. In the present study we have used small angle neutron scattering to study mixed micelles of lauryldimethyl amine oxide (LDAO; hydrogenated and deuterated) and heptane-1,2,3-triol (HP). Our results show that with increasing overall HP concentrations mixed LDAO/HP micelles of decreasing mass and radius are formed. The composition of these micelles has been determined. HP thus may decrease the size of the surfactant belt around a protein before crystallisation by insertion into a host micelle. As HP is a 'small amphiphile' compared to the surfactants used for solubilization of membrane proteins, the curvature of the host micelle will be increased by its insertion.
Crystallizing membrane proteins remains a challenging endeavor despite the increasing number of membrane protein structures solved by X-ray crystallography. The critical factors in determining the success of the crystallization experiments are the purification and preparation of membrane protein samples. Moreover, there is the added complication that the crystallization conditions must be optimized for use in the presence of detergents although the methods used to crystallize most membrane proteins are, in essence, straightforward applications of standard methodologies for soluble protein crystallization. The roles that detergents play in stability and aggregation of membrane proteins as well as the colloidal properties of the protein-detergent complexes need to be appreciated and controlled before and during the crystallization trials. All X-ray quality crystals of membrane proteins were grown from preparations of detergent-solubilized protein, where the heterogeneous natural lipids from the membrane have been replaced by a homogeneous detergent environment. It is the preparation of such monodisperse, isotropic solutions of membrane proteins that has allowed the successful application of the standard crystallization methods routinely used on soluble proteins. In this review, the issues of protein purification and sample preparation are addressed as well as the new refinements in crystallization methodologies for membrane proteins. How the physical behavior of the detergent, in the form of micelles or protein-detergent aggregates, affects crystallization and the adaptation of published protocols to new membrane protein systems are also addressed. The general conclusion is that many integral membrane proteins could be crystallized if pure and monodisperse preparations in a suitable detergent system can be prepared.
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