Forces and factors that contribute to the structural stability of membrane proteins

While a considerable amount of literature deals with the structural energetics of water-soluble proteins, relatively little is known about the forces that determine the stability of membrane proteins. Similarly, only a few membrane protein structures are known at atomic resolution, although new structures have recently been described. In this article, we review the current knowledge about the structural features of membrane proteins. We then proceed to summarize the existing literature regarding the thermal stability of bacteriorhodopsin, cytochrome-c oxidase, the band 3 protein, Photosystem II and porins. We conclude that a fundamental difference between soluble and membrane proteins is the high thermal stability of intrabilayer secondary structure elements in membrane proteins. This property manifests itself as incomplete unfolding, and is reflected in the observed low enthalpies of denaturation of most membrane proteins. By contrast, the extramembranous parts of membrane proteins may behave much like soluble proteins. A brief general account of thermodynamics factors that contribute to the stability of water soluble and membrane proteins is presented.     

用神经网络法预测膜蛋白RH和BR的二级结构

膜蛋白的结构预测目前还十分困难.本文以两个典型的膜蛋白为选材,用神经网络方法对其二级结构做了预测.预测结果与实验资料的符合率与该方法用于球蛋白时的结果一致,因此是较好的.鉴于目前还没有人将此方法用于膜蛋白的结构预测.我们拟进一步发展此方法.     

Fragment-based phase extension for three-dimensional structure determination of membrane proteins by electron crystallography

In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.     

A simple statistical method for discriminating outer membrane proteins with better accuracy

MOTIVATION: Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important task both for identifying outer membrane proteins from genomic sequences and for the successful prediction of their secondary and tertiary structures. RESULTS: We have systematically analyzed the amino acid composition of globular proteins from different structural classes and outer membrane proteins. We found that the residues, Glu, His, Ile, Cys, Gln, Asn and Ser, show a significant difference between globular and outer membrane proteins. Based on this information, we have devised a statistical method for discriminating outer membrane proteins from other globular and membrane proteins. Our approach correctly picked up the outer membrane proteins with an accuracy of 89% for the training set of 337 proteins. On the other hand, our method has correctly excluded the globular proteins at an accuracy of 79% in a non-redundant dataset of 674 proteins. Furthermore, the present method is able to correctly exclude alpha-helical membrane proteins up to an accuracy of 80%. These accuracy levels are comparable to other methods in the literature, and this is a simple method, which could be used for dissecting outer membrane proteins from genomic sequences. The influence of protein size, structural class and specific residues for discrimination is discussed.     

Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures

Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield high-resolution structures.     

Non-covalent binding of membrane lipids to membrane proteins

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function. However only relatively recently has high resolution structure determination of membrane proteins progressed to the point of providing atomic level structure of lipid binding sites on membrane proteins. Analysis of X-ray diffraction, electron crystallography and NMR data over 100 specific lipid binding sites on membrane proteins. These data demonstrate tight lipid binding of both phospholipids and cholesterol to membrane proteins. Membrane lipids bind to membrane proteins by their headgroups, or by their acyl chains, or binding is mediated by the entire lipid molecule. When headgroups bind, binding is stabilized by polar interactions between lipid headgroups and the protein. When acyl chains bind, van der Waals effects dominate as the acyl chains adopt conformations that complement particular sites on the rough protein surface. No generally applicable motifs for binding have yet emerged. Previously published biochemical and biophysical data link this binding with function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na(+)-citrate symporter CitS as an example

The determination of 3D structures of membrane proteins is still extremely difficult. The co-crystallization with specific binding proteins may be an important aid in this process, as these proteins provide rigid, hydrophilic surfaces for stable protein-protein contacts. Also, the conformational homogeneity of the membrane protein may be increased to obtain crystals suitable for high resolution structures. Here, we describe the efficient generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) as specific binding molecules for membrane proteins. We used both phage display and ribosome display to select DARPins in vitro that are specific for the detergent-solubilized Na(+)-citrate symporter CitS of Klebsiella pneumoniae. Compared to classical hybridoma technology, the in vitro selection systems allow a much better control of the structural integrity of the target protein and allow the use of other protein classes in addition to recombinant antibodies. We also compared the selected DARPins to a Fab fragment previously selected by phage display and demonstrate that different epitopes are recognized, unique to each class of binding molecules. Therefore, the use of several classes of binding molecules will make suitable crystal formation and the determination of their 3D structure more likely.     

The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution

We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data. This is the largest protein structure thus far determined by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10. Sequence-specific 1H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332-6341]. Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data.(ABSTRACT TRUNCATED AT 400 WORDS)     

Site-specific examination of secondary structure and orientation determination in membrane proteins: the peptidic (13)C=(18)O group as a novel infrared probe

Detailed site-specific information can be exceptionally useful in structural studies of macromolecules in general and proteins in particular. Such information is usually obtained from spectroscopic studies using a label/probe that can reflect on particular properties of the protein. A suitable probe must not modify the native properties of the protein, and should yield interpretable structural information, as is the case with isotopic labels used by Fourier transform infrared (FTIR) spectroscopy. In particular, 1-(13)C=(18)O labels have been shown to relay site-specific secondary structure and orientational information, although limited to small peptides. The reason for this limitation is the high natural abundance of (13)C and the lack of baseline resolution between the main amide I band and the isotope-edited peak. Herein, we dramatically extend the utility of isotope edited FTIR spectroscopy to proteins of virtually any size through the use of a new 1-(13)C=(18)O label. The double-isotope label virtually eliminates any contribution from natural abundance (13)C. More importantly, the isotope-edited peak is further red-shifted (in accordance with ab initio Hartree-Fock calculations) and is now completely baseline resolved from the main amide I band. Taken together, this new label enables determination of site specific secondary structure and orientation in proteins of virtually any size. Even in small peptides 1-(13)C=(18)O is far preferable as a label in comparison to 1-(13)C=(18)O since it enables analysis without the need for any deconvolution or peak fitting procedures. Finally, the results obtained herein represent the first stage in the application of site-directed dichroism to the structural elucidation of polytopic membrane proteins.     

Native secondary structure topology has near minimum contact energy among all possible geometrically constrained topologies

Secondary structure topology in this article refers to the order and the direction of the secondary structures, such as helices and strands, with respect to the protein sequence. Even when the locations of the secondary structure Cα atoms are known, there are still (N!2^N)(M!2^M) different possible topologies for a protein with N helices and M strands. This work explored the question if the native topology is likely to be identified among a large set of all possible geometrically constrained topologies through an evaluation of the residue contact energy formed by the secondary structures, instead of the entire chain. We developed a contact pair specific and distance specific multiwell function based on the statistical characterization of the side chain distances of 413 proteins in the Protein Data Bank. The multiwell function has specific parameters to each of the 210 pairs of residue contacts. We illustrated a general mathematical method to extend a single well function to a multiwell function to represent the statistical data. We have performed a mutation analysis using 50 proteins to generate all the possible geometrically constrained topologies of the secondary structures. The result shows that the native topology is within the top 25% of the list ranked by the effective contact energies of the secondary structures for all the 50 proteins, and is within the top 5% for 34 proteins. As an application, the method was used to derive the structure of the skeletons from a low resolution density map that can be obtained through electron cryomicroscopy. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Effects of intracellular sodium and potassium iontophoresis on membrane potentials and resistances in toad urinary bladder

Conclusion The elastic X-ray/neutron diffraction techniques described in this review are currently capable of providing substantial information concerning the time-averaged structures and intermolecular ordering of molecular components within a dynamic membrane structure. In addition, the time resolution of the elastic X-ray diffraction technique, afforded by intense synchrotron and laser plasma X-ray sources, now permits this structural information to be obtained over a range of time scales from nanoseconds to milliseconds and upwards following an excitation of the membrane system. This time-averaged and time-resolved structural information may provide considerable insight into structure-function relationships in biological membranes and, especially when combined with structural information on the membrane proteins involved at atomic resolution, may provide this insight at the atomic level.     

Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase

Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo₃ oxidase. The optimized protocol for expression and purification yields ∼5 mg of the enzymatically active, uniformly ¹³C,¹⁵N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The ¹³C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo₃ oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly ¹³C,¹⁵N-labeled membrane protein. Heather L. Frericks and Lai Lai Yap contributed equally to this work.     

Comparison of NMR and crystal structures of membrane proteins and computational refinement to improve model quality

Membrane proteins are challenging to study and restraints for structure determination are typically sparse or of low resolution because the membrane environment that surrounds them leads to a variety of experimental challenges. When membrane protein structures are determined by different techniques in different environments, a natural question is “which structure is most biologically relevant?” Towards answering this question, we compiled a dataset of membrane proteins with known structures determined by both solution NMR and X‐ray crystallography. By investigating differences between the structures, we found that RMSDs between crystal and NMR structures are below 5 Å in the membrane region, NMR ensembles have a higher convergence in the membrane region, crystal structures typically have a straighter transmembrane region, have higher stereo‐chemical correctness, and are more tightly packed. After quantifying these differences, we used high‐resolution refinement of the NMR structures to mitigate them, which paves the way for identifying and improving the structural quality of membrane proteins.     

Sequence and conformational preferences at termini of α‐helices in membrane proteins: Role of the helix environment

α‐helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α‐helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C‐termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α‐helices in a high‐resolution dataset of integral α‐helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C‐termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near‐helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420–3436. © 2014 Wiley Periodicals, Inc.     

A simple method for correction of circular dichroism spectra obtained from membrane-containing samples

Circular dichroism (CD) spectroscopy is an important technique in structural biology for examining folding and conformational changes of proteins in solution. However, the use of CD spectroscopy in a membrane medium (and also in a nonhomogeneous medium) is limited by (i) high light scattering and (ii) differential scattering of incident left and right circularly polarized light, especially at shorter wavelengths (<200 nm). We report a novel methodology for estimating the distortion of CD spectra caused by light scattering for membrane-bound peptides and proteins. The method is applied to three proteins with very different secondary structures to illustrate the limits of its capabilities when calibrated with a simple soluble peptide ([Ac]ANLKALEAQKQKEQRQAAEELANAK[OH], standard peptide) with a balanced secondary structure. The method with this calibration standard was quite successful in estimating α-helix but more limited when it comes to proteins with very high β-sheet or β-turn content.     

The Basic Protein of CNS Myelin: Its Structure and Ligand Binding

Consideration of the evidence presented in this review leads to the following conclusions: (a) Isolated MBP in aqueous solution has little ordered secondary or tertiary structure. (b) In this state, the protein can associate with a wide range of hydrophobic and amphiphilic compounds, these interactions involving limited sections of the protein. (c) The strength of binding to bilayers and the accompanying conformational changes in the protein are greatest for systems containing acidic lipids, presumably because of the involvement of ionic interactions. (d) When bound to bilayers of acidic lipids, MBP will have substantially more ordered secondary structure than it manifests in aqueous solution, and it is likely to be oligomeric (possibly hexameric). (e) MBP does affect the organization of lipid aggregates. It influences strongly the separation of bilayers in multilayers of purified lipids, and at present this must be viewed as its prime role within myelin. The greatest impediment to our understanding of MBP is the lack of an assayable biological activity. In contrast to the situation with enzymes, for example, we have no functional test for changes in protein structure or changes accompanying interactions with other molecules. Current evidence suggests that the protein has a structural role within myelin and that its own three-dimensional structure is strongly dependent on the molecules with which it is associated. If this picture is correct, studies of the isolated protein or of the protein in reconstituted lipid systems may yield, at best, a rough guide to the structure within its biological environment. Further clarification of the structure and function of MBP may have to await development of more powerful techniques for studying proteins bound to large molecular aggregates, such as lipid bilayers. The paucity of generally applicable methods is reflected in the fact that even low resolution structures are known for only a handful of intrinsic membrane proteins, and even more limited information exists for proteins associated with membrane surfaces. However, the increasing use of a combination of electron microscopy and diffraction on two-dimensional arrays of proteins formed on lipid bilayers (Henderson et al., 1990) offers the hope that it may not be too long before it will be possible to study at moderate resolution the three-dimensional structure of MBP bound to a lipid membrane.     

The effect of ATP on the cell membrane structure]

ATP influence on the structure of plasma membranes thymocytes of cattle was studied. Fluorescence anisotropy of tryptophan residues of membrane proteins, fluorescence anisotropy of 3-methoxybenzanetron and fluorescence intensity of 1-anilinonaphthalene-8-sulphonate were determined. Changes of tryptophan fluorescence anisotropy and of ANS fluorescence intensity were established. It is supposed that the observed changes are connected with the change of membrane proteins structure and plasma membrane charge.     

Prediction of protein secondary structures from conformational biases

We use LINUS (the "Local Independently Nucleated Units of Structure"), a procedure developed by Srinivasan and Rose, to provide a physical interpretation of and predict the secondary structures of proteins. The secondary structure type at a given site is identified by the largest conformational bias during short simulations. We examine the rate of successful prediction as a function of temperature and the interaction window. At high temperatures, there is a large propensity for the establishment of beta-strands whereas alpha-helices appear only when the temperature is lower than a certain threshold value. It is found that there exists an optimal temperature at which the correct secondary structures are predicted most accurately. We find that this temperature is close to the peak temperature of the specific heat. Changing the interaction window or carrying out longer simulations approaching equilibrium lead to little change in the optimal success rate. Our findings are in accord with the observation by Srinivasan and Rose that the secondary structures are mainly determined by local interactions and appear in the early stage of folding.     

High-efficiency screening of monoclonal antibodies for membrane protein crystallography

Determination of crystal structures of membrane proteins is often limited by difficulties obtaining crystals diffracting to high resolution. Co-crystallization with Fab fragments of monoclonal antibodies has been reported to improve diffraction of membrane proteins crystals. However, it is not simple to generate useful monoclonal antibodies for membrane protein crystallography. In this report, we present an optimized process for efficient screening from immunization to final validation of monoclonal antibody for membrane protein crystallography.     

Bilayer structure and physical dynamics of the cytochrome b5 dimyristoylphosphatidylcholine interaction.

Cytochrome b5 is a microsomal membrane protein which provides reducing potential to delta 5-, delta 6-, and delta 9-fatty acid desaturases through its interaction with cytochrome b5 reductase. Low angle x-ray diffraction has been used to determine the structure of an asymmetrically reconstituted cytochrome b5:DMPC model membrane system. Differential scanning calorimetry and fluorescence anisotropy studies were performed to examine the bilayer physical dynamics of this reconstituted system. These latter studies allow us to constrain structural models to those which are consistent with physical dynamics data. Additionally, because the nonpolar peptide secondary structure remains unclear, we tested the sensitivity of our model to different nonpolar peptide domain configurations. In this modeling approach, the nonpolar peptide moiety was arranged in the membrane to meet such chemically determined criteria as protease susceptibility of carboxyl- and amino-termini, tyrosine availability for pH titration and tryptophan 109 location, et cetera. In these studies, we have obtained a reconstituted cytochrome b5:DMPC bilayer structure at approximately 6.3 A resolution and conclude that the nonpolar peptide does not penetrate beyond the bilayer midplane. Structural correlations with calorimetry, fluorescence anisotropy and acyl chain packing data suggest that asymmetric cytochrome b5 incorporation into the bilayer increases acyl chain order. Additionally, we suggest that the heme peptide:bilayer interaction facilitates a discreet heme peptide orientation which would be dependent upon phospholipid headgroup composition.