FIGURE 6 - uploaded by Sarah L Rouse
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Destabilizing interactions of zwitterionic detergent molecules with BM2 protein during dehydration. (Upper) The change in conformation of the BM2-DDM complex at ~0.62 ms (see Fig. 5) correlates with extraction of a water molecule (green and white) from the central pore, mediated by interactions with the headgroup of a DHPC molecule. The water and DHPC molecule are shown in spacefilling format, and the protein (yellow) in cartoon format. (Lower) Destabilizing interactions of DPC detergent molecules similar to those depicted above, with the C terminus of BM2 are observed, in which a single detergent molecule gradually becomes lodged between two of the transmembrane helices, interacting with water molecules and remaining within the pore for the duration of the simulation. Water molecules within 4 A ˚ of this His 27 residue are shown as a green surface.
Source publication
Molecular dynamics simulations have been used to characterize the effects of transfer from aqueous solution to a vacuum to inform our understanding of mass spectrometry of membrane-protein-detergent complexes. We compared two membrane protein architectures (an α-helical bundle versus a β-barrel) and two different detergent types (phosphocholines ve...
Contexts in source publication
Context 1
... hydrophilic residues relative to packing trends for the PDC in water. These changes can be quantified as the degree of detergent headgroup clustering in each simulation (Fig. S6). There is a clear decrease in minimum distances between DHPC and DPC headgroups in vacuum compared to solution. In contrast, in the BM2-DDM PDC, there was very little change in detergent headgroup clustering upon the transition to ...
Context 2
... of the protein conformational changes. In the case of BM2-DHPC, the decrease in solvation leads to increased electrostatic interactions between the zwitterionic DHPC headgroups and protein residues. Thus, the phosphocholine headgroups may penetrate between helices to interact with water molecules within the central pore of the BM2 helix bundle (Fig. 6), leading to extrusion of the water molecule laterally through the helix bundle. In the BM2-DPC simula- tion, similar destabilizing interactions between detergent headgroups and the protein occur, in which a single deter- gent molecule adopts a nonnative orientation and projects into the pore of the BM2 channel, leading to loss of the ...
Context 3
... to extrusion of the water molecule laterally through the helix bundle. In the BM2-DPC simula- tion, similar destabilizing interactions between detergent headgroups and the protein occur, in which a single deter- gent molecule adopts a nonnative orientation and projects into the pore of the BM2 channel, leading to loss of the solution conformation (Fig. 6). Furthermore, as the simula- tion progresses the protein becomes increasingly expelled from the detergent, leading to greater distortions of the solution ...
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In this study, the natural deep eutectic solvents (NADESs) based on trehalose and choline chloride have been prepared to enhance the protein thermostability. The results of fourier transform infrared spectroscopy and ¹H nuclear magnetic resonance spectroscopy suggested that there were intensive hydrogen-bonding interactions between trehalose and ch...
Citations
... 36,37 Similar changes can be observed in MD simulations of desolvated protein-detergent complexes: contacts between detergent head-groups and the protein increase, while the hydrophobic alkyl chains extend away from the surface into the vacuum. 38,39 Additionally, the inversion process is promoted by thermal activation, which is typically used to remove detergent from the protein ions during mass spectrometry analysis. 37 In the results we present here, the detergent head-groups have the most pronounced effect on the nal charge of a membrane protein, which indicates that the head groups are the most likely sites for protonation on a detergent molecule. ...
Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion-ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.
... Two proteins and a peptide were initially prepared in HBS buffer containing [10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% n-dodecyl-β-D-maltoside (DDM)]. DDM is known to be an effective membrane protein stabilizing detergent [38][39][40]. The CM5 sensor chip surface was first activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxy succinimide (NHS) mixture using a Biacore 8K instrument (Cytiva). ...
The Sigma‐2 receptor (S2R) (a.k.a TMEM97) is an important endoplasmic reticular protein involved in cancer, cholesterol processing, cell migration, and neurodegenerative diseases, including Niemann–Pick Type C. While several S2R pharmacologic agents have been discovered, its recent (2017) cloning has limited biological investigation, and no endogenous ligands of the S2R are known. Histatins are a family of endogenous antimicrobial peptides that have numerous important effects in multiple biological systems, including antifungal, antibacterial, cancer pathogenesis, immunomodulation, and wound healing. Histatin‐1 (Hst1) has important roles in epithelial wound healing and cell migration, and is the primary wound healing agent in saliva. Little is understood about the downstream machinery that underpins the effects of histatins, and no mammalian receptor is known to date. In this study, we show, using biophysical methods and functional assays, that Hst1 is an endogenous ligand for S2R and that S2R is a mammalian receptor for Hst1.
... In order to minimize irreversible destructive changes in the RC protein during the formation of films, the RCs were suspended in a buffer containing DM. The micelles of this mild nonionic detergent have a pronounced capability to maintain the structure of membrane proteins, in particular during the phase transition from solution to vacuum [24,25]. The stabilizing effect of DM on Rba. ...
The photoinduced charge separation in QB-depleted reaction centers (RCs) from Rhodobacter sphaeroides R-26 in solid air-dried and vacuum-dried (~10⁻² Torr) films, obtained in the presence of detergent n-dodecyl-β-D-maltoside (DM), is characterized using ultrafast transient absorption spectroscopy. It is shown that drying of RC-DM complexes is accompanied by reversible blue shifts of the ground-state absorption bands of the pigment ensemble, which suggest that no dehydration-induced structural destruction of RCs occurs in both types of films. In air-dried films, electron transfer from the excited primary electron donor P⁎ to the photoactive bacteriopheophytin HA proceeds in 4.7 ps to form the P⁺HA⁻ state with essentially 100% yield. P⁺HA⁻ decays in 260 ps both by electron transfer to the primary quinone QA to give the state P⁺QA⁻ (87% yield) and by charge recombination to the ground state (13% yield). In vacuum-dried films, P⁎ decay is characterized by two kinetic components with time constants of 4.1 and 46 ps in a proportion of ~55%/45%, and P⁺HA⁻ decays about 2-fold slower (462 ps) than in air-dried films. Deactivation of both P⁎ and P⁺HA⁻ to the ground state effectively competes with the corresponding forward electron-transfer reactions in vacuum-dried RCs, reducing the yield of P⁺QA⁻ to 68%. The results are compared with the data obtained for fully hydrated RCs in solution and are discussed in terms of the presence in the RC complexes of different water molecules, the removal/displacement of which affects spectral properties of pigment cofactors and rates and yields of the electron-transfer reactions.
... Apparently, no data on the effect of detergents on the membrane proteins in dry films in vacuum are available in the literature. However, there are extensive data for the complexes of membrane proteins with various detergents in vacuum obtained in mass spectrometry studies [10,11] and by using molecu lar dynamics methods [35]. It was found that DM micelles have a pronounced ability to maintain the integrity of membrane proteins during their transition from solution to vacuum in a mass spectrometer [10]. ...
... It was found that DM micelles have a pronounced ability to maintain the integrity of membrane proteins during their transition from solution to vacuum in a mass spectrometer [10]. Recent molecular dynamics simulations have demon strated that the stabilizing effect of DM on the structure of an α helical membrane protein in vacuum is apparent ly related to the preservation of the DM micelle structure characteristic of the solution due to the formation of additional hydrogen bonds between maltose residues [35]. At the same time, vacuum dehydration of complexes of zwitterionic detergents with α helical membrane proteins led to the micelle inversion and partial exposure of pro tein surface in vacuum, which was accompanied by noticeable changes in the secondary and tertiary protein structure [35]. ...
... Recent molecular dynamics simulations have demon strated that the stabilizing effect of DM on the structure of an α helical membrane protein in vacuum is apparent ly related to the preservation of the DM micelle structure characteristic of the solution due to the formation of additional hydrogen bonds between maltose residues [35]. At the same time, vacuum dehydration of complexes of zwitterionic detergents with α helical membrane proteins led to the micelle inversion and partial exposure of pro tein surface in vacuum, which was accompanied by noticeable changes in the secondary and tertiary protein structure [35]. LDAO contains a highly polar amine oxide group and its properties are more similar to that of ionic alkyl detergents rather than non ionic detergents [36]. ...
Photochemical reaction centers (RCs) of photosynn thetic bacteria are integral membrane pigment-protein complexes that convert the energy of light quanta into electrochemical energy of separated charges through a series of fast electron transfer reactions [1]. The RC of the purple bacterium Rhodobacter sphaeroides contains three protein subunits (L, M, and H), two of which (L and M) bind cofactors that form two quasiisymmetrical electron transfer branches [2, 3] often designated as A and B. The branches share a dimer of excitonically coupled bacterioo chlorophyll (BChl) molecules that serves as a primary electron donor (P); each of them includes a molecule of monomeric BChl (B A or B B , respectively), a bacterioo pheophytin (BPheo) molecule (H A or H B), and a ubiquinone molecule (Q A or Q B). Only the A branch is active in the primary charge separation [1]. XXray studies revealed that RC crystals also contain a large number of bound water molecules, some of which are functionally active [3, 4]. As natural nanoscale energyytransforming structures, bacterial RCs are characterized by an extremely high (almost 100%) quantum yield of charge separation that is achieved as a result of fast (~200 ps) "cascade" electron transfer from the singlettexcited prii mary electron donor P* through the shorttlived ion pairs P + B A-and P + H A-to the primary quinone acceptor Q A with
... Native MS affords the ability to analyze intact protein complexes and preserve noncovalent interactions in the mass spectrometer for analysis (28)(29)(30)(31)(32). Although MS has been applied to soluble proteins for nearly three decades (31), recent advances have led to the ability to preserve noncovalent interactions and maintain intact, folded membrane proteins in the gas phase, providing invaluable information on subunit stoichiometry, nucleotide, drug, peptide, and lipid binding (7,(33)(34)(35)(36)(37). Importantly, the detergent micelle protects membrane proteins from solution into the mass spectrometer (38,39). In the case of membrane protein−lipid complexes, minimal activation is applied to gently remove the detergent micelle while preserving both native-like structure and noncovalently bound lipids (7,(40)(41)(42)(43)(44)(45). ...
Significance
The diverse environment of cellular membranes presents unique challenges in deciphering the roles that lipids play in modulating membrane protein structure and function. Here, we developed a native mass spectrometry approach to monitor binding of different lipid types to membrane proteins. We discovered that specific lipid−protein interactions can allosterically modulate the binding of lipids of different types. We also determined the structure of AmtB bound to cardiolipin, and mutation of residues involved in binding this lipid abolishes the observed allosteric effect. Our findings are of particular significance as they contribute to our general knowledge of how lipids modulate protein structure and function and how membrane proteins may recruit, through allostery, their own lipid microenvironment.
... Specifically, it would be relevant to understand how lipids can still be bound to the protein after micelle destruction in the gas phase (Figure 2A). By steering MD simulations, Rouse et al. have shown that a MP-micelle complex in solution can be transferred to the gas phase, observing conformational changes in the micelle structure (Rouse et al., 2013). Hall et al performed MD simulations of SAP soluble protein in vacuum at increasing temperatures, from 300 to 800 • K, in order to represent increasing activation energy in Ion Mobility-Mass Spectrometry (IM-MS) experiments, which resulted in a good correlation between the experimental and theoretical values (Hall et al., 2012a). ...
Membrane proteins represent a challenging family of macromolecules, particularly related to the methodology aimed at characterizing their three-dimensional structure. This is mostly due to their amphipathic nature as well as requirements of ligand bindings to stabilize or control their function. Recently, Mass Spectrometry (MS) has become an important tool to identify the overall stoichiometry of native-like membrane proteins complexed to ligand bindings as well as to provide insights into the transport mechanism across the membrane, with complementary information coming from X-ray crystallography. This perspective article emphasizes MS findings coupled with X-ray crystallography in several membrane protein lipid complexes, in particular transporters, ion channels and molecular machines, with an overview of techniques that allows a more thorough structural interpretation of the results, which can help us to unravel hidden mysteries on the membrane protein function.
... D espite the broad array of essential functions executed by membrane proteins (MPs), high resolution structural data for this class of proteins are lacking compared with their water-soluble counterparts. Electrospray ionization-mass spectrometry (ESI-MS) is emerging as an invaluable method with which to study MPs, allowing them to be transferred to the gas phase in a native-like state from a suitable amphiphile, and affording insights into MP mass, conformation, and small molecule binding [1][2][3][4][5][6][7][8]. n-Dodecyl-β-maltoside (DDM) is a frequently used detergent for such analyses [2][3][4][5] but recently the use of amphipols [1,2,8,9] and other amphiphiles [6,10] have been reported. ...
... Electrospray ionization-mass spectrometry (ESI-MS) is emerging as an invaluable method with which to study MPs, allowing them to be transferred to the gas phase in a native-like state from a suitable amphiphile, and affording insights into MP mass, conformation, and small molecule binding [1][2][3][4][5][6][7][8]. n-Dodecyl-β-maltoside (DDM) is a frequently used detergent for such analyses [2][3][4][5] but recently the use of amphipols [1,2,8,9] and other amphiphiles [6,10] have been reported. Native MS can also be used in conjunction with other gas phase techniques, such as ion mobility spectrometry (IMS) [11][12][13][14][15] and collision induced dissociation and collision induced unfolding (CID/CIU) [16,17] to probe the structure and topology of MPs and their complexes. ...
Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles. Using quantitative mass spectrometry analyses, we show that fast photochemical oxidation reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of additional intermolecular contacts.
... n-Dodecyl-b-D-maltoside n-Dodecyl-b-D-maltoside (DDM) is a non-ionic detergent with an aliphatic 12-carbon chain (Table I and Figure 1A), which tends to disrupt lipid-lipid and lipid-protein interactions but not protein-protein interactions. The mild and non-denaturing properties of DDM make it a commonly used detergent for the extraction and purification of membrane proteins and for solubilization in experimental studies of their structure, dynamics and function (Ward et al., 2000, Seddon et al., 2004, Arnold & Linke, 2008, Pagliano et al., 2012, Rouse et al., 2013. DDM tends to retain the native structure and functional activity of membrane proteins to a greater extent than any other detergents (Alexandrov et al., 2008). ...
Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilisation of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilisation of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilise membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilised on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesised with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.
... n-Dodecyl-b-D-maltoside n-Dodecyl-b-D-maltoside (DDM) is a non-ionic detergent with an aliphatic 12-carbon chain (Table I and Figure 1A), which tends to disrupt lipid-lipid and lipid-protein interactions but not protein-protein interactions. The mild and non-denaturing properties of DDM make it a commonly used detergent for the extraction and purification of membrane proteins and for solubilization in experimental studies of their structure, dynamics and function (Ward et al., 2000, Seddon et al., 2004, Arnold & Linke, 2008, Pagliano et al., 2012, Rouse et al., 2013. DDM tends to retain the native structure and functional activity of membrane proteins to a greater extent than any other detergents (Alexandrov et al., 2008). ...
This work reports the first synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d39-DDM). DDM is a mild non-ionic detergent often used in the extraction and purification of membrane proteins and for solubilising them in experimental studies of their structure, dynamics and binding of ligands. We required d39-DDM for solubilising large α-helical membrane proteins in samples for [15N-1H]TROSY NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d39-DDM used d7-D-glucose and d25-n-dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α-configuration, were coupled together with an α(1→4) glycosidic bond to give maltose, which was then coupled with n-dodecanol by its substitution of a C1 bromine in the α-configuration to give DDM. 1H NMR spectra were used to confirm a high level of deuteration in the synthesised d39-DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52 kDa sugar transport protein solubilised in DDM.
... Examples of this include MD simulations of crystal packing in OmpA 8 and of the behavior of protein−detergent complexes (PDCs) in mass spectrometry experiments. 9 Simulations of PDCs pose a number of additional challenges as the time scales required to fully sample the process of micelle formation are not readily accessible by conventional atomistic (AT) simulation, while reduced representations (e.g., coarsegrained simulations) may not capture all of the details of the process. 10 However, these limitations may be overcome by a variety of methods, including multiscale approaches, 10 and by averaging over multiple simulations. ...
Structural studies of membrane proteins have highlighted the likely influence of membrane mimetic environments (i.e. lipid bilayers vs. detergent micelles) on the conformation and dynamics of small α-helical membrane proteins. We have used molecular dynamics simulations to compare the conformational dynamics of BM2 (a small α-helical protein from the membrane of influenza B) in a model phospholipid bilayer environment with its behavior in protein-detergent complexes with either the zwitterionic detergent dihexanoylphosphatidylcholine (DHPC) or the non-ionic detergent dodecylmaltoside (DDM). We find that DDM more closely resembles the lipid bilayer in terms of its interaction with the protein, whilst the short-tailed DHPC molecule forms 'non-physiological' interactions with the protein termini. We find that the intrinsic micelle properties of each detergent are conserved upon formation of the protein-detergent complex. This implies that simulations of detergent micelles may be used to help select optimal conditions for experimental studies of membrane proteins.
