Residues In Membrane Proteins Research Articles

Automated Fragmentation Quantum Mechanical Calculation of 15N and 13C Chemical Shifts in a Membrane Protein.

In this work, we developed an accurate and cost-effective automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) method to calculate the chemical shifts of 15N and 13C of membrane proteins. The convergence of the AF-QM/MM method was tested using Krokinobacter eikastus rhodopsin 2 as a test case. When the distance threshold of the QM region is equal to or larger than 4.0 Å, the results of the AF-QM/MM calculations are close to convergence. In addition, the effects of selected density functionals, basis sets, and local chemical environment of target atoms on the chemical shift calculations were systematically investigated. Our results demonstrate that the predicted chemical shifts are more accurate when important environmental factors including cross-protomer interactions, lipid molecules, and solvent molecules are taken into consideration, especially for the 15N chemical shift prediction. Furthermore, with the presence of sodium ions in the environment, the chemical shift of residues, retinal, and retinal Schiff base are affected, which is consistent with the results of the solid-state nuclear magnetic resonance (NMR) experiment. Upon comparing the performance of various density functionals (namely, B3LYP, B3PW91, M06-2X, M06-L, mPW1PW91, OB95, and OPBE), the results show that mPW1PW91 is a suitable functional for the 15N and 13C chemical shift prediction of the membrane proteins. Meanwhile, we find that the improved accuracy of the 13Cβ chemical shift calculations can be achieved by the employment of the triple-ζ basis set. However, the employment of the triple-ζ basis set does not improve the accuracy of the 15N and 13Cα chemical shift calculations nor does the addition of a diffuse function improve the overall prediction accuracy of the chemical shifts. Our study also underscores that the AF-QM/MM method has significant advantages in predicting the chemical shifts of key ligands and nonstandard residues in membrane proteins than most widely used empirical models; therefore, it could be an accurate computational tool for chemical shift calculations on various types of biological systems.

The Functional Significance of Hydrophobic Residue Distribution in Bacterial Beta-Barrel Transmembrane Proteins.

β-barrel membrane proteins have several important biological functions, including transporting water and solutes across the membrane. They are active in the highly hydrophobic environment of the lipid membrane, as opposed to soluble proteins, which function in a more polar, aqueous environment. Globular soluble proteins typically have a hydrophobic core and a polar surface that interacts favorably with water. In the fuzzy oil drop (FOD) model, this distribution is represented by the 3D Gauss function (3DG). In contrast, membrane proteins expose hydrophobic residues on the surface, and, in the case of ion channels, the polar residues face inwards towards a central pore. The distribution of hydrophobic residues in membrane proteins can be characterized by means of 1–3DG, a complementary 3D Gauss function. Such an analysis was carried out on the transmembrane proteins of bacteria, which, despite the considerable similarities of their super-secondary structure (β-barrel), have highly differentiated properties in terms of stabilization based on hydrophobic interactions. The biological activity and substrate specificity of these proteins are determined by the distribution of the polar and nonpolar amino acids. The present analysis allowed us to compare the ways in which the different proteins interact with antibiotics and helped us understand their relative importance in the development of the resistance mechanism. We showed that beta barrel membrane proteins with a hydrophobic core interact less strongly with the molecules they transport.

Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method.

Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.

Phactr3/scapinin, a member of protein phosphatase 1 and actin regulator (phactr) family, interacts with the plasma membrane via basic and hydrophobic residues in the N-terminus.

Proteins that belong to the protein phosphatase 1 and actin regulator (phactr) family are involved in cell motility and morphogenesis. However, the mechanisms that regulate the actin cytoskeleton are poorly understood. We have previously shown that phactr3, also known as scapinin, localizes to the plasma membrane, including lamellipodia and membrane ruffles. In the present study, experiments using deletion and point mutants showed that the basic and hydrophobic residues in the N-terminus play crucial roles in the localization to the plasma membrane. A BH analysis (http://helixweb.nih.gov/bhsearch) is a program developed to identify membrane-binding domains that comprise basic and hydrophobic residues in membrane proteins. We applied this program to phactr3. The results of the BH plot analysis agreed with the experimentally determined region that is responsible for the localization of phactr3 to the plasma membrane. In vitro experiments showed that the N-terminal itself binds to liposomes and acidic phospholipids. In addition, we showed that the interaction with the plasma membrane via the N-terminal membrane-binding sequence is required for phactr3-induced morphological changes in Cos7 cells. The membrane-binding sequence in the N-terminus is highly conserved in all members of the phactr family. Our findings may provide a molecular basis for understanding the mechanisms that allow phactr proteins to regulate cell morphogenesis.

How Do Membrane Transporters Sense pH? The Case of the Mitochondrial ADP–ATP Carrier

The activity of many membrane proteins depends markedly on the pH. Pinpointing the amino acids forming the pH sensor domains of these proteins remains challenging for current experimental techniques. Combining molecular dynamics simulations and pKa predictions with in vitro transport assays, we have revealed the molecular basis of the pH dependence of the mitochondrial carrier mediating the exchanges of ADP3– and ATP4– across the inner mitochondrial membrane. We have demonstrated that the transport activity of this mitochondrial carrier depends on the protonation state of both the substrate and a unique, highly conserved residue of the protein. The original strategy proposed here offers a convenient framework for identifying pH-sensitive residues in membrane proteins in such cases where one single amino acid is involved. Our findings are envisioned to help toward the rational design of active compounds ranging from drugs to biosensors.

Bioinformatics approaches for functional annotation of membrane proteins

Membrane proteins perform diverse functions in living organisms such as transporters, receptors and channels. The functions of membrane proteins have been investigated with several computational approaches, such as developing databases, analyzing the structure-function relationship and establishing algorithms to discriminate different type of membrane proteins. However, compilation of bioinformatics resources for the functions of membrane proteins is not well documented compared with their structural aspects. In this comprehensive review, we elaborately focus on three aspects of membrane protein functions: (i) databases for different types of membrane proteins based on their functions including transporters, receptors and ion channels, annotated functional data for genomes, as well as functionally important amino acid residues in membrane proteins obtained from experimental data, (ii) analysis of membrane protein functions based on their structures, motifs, amino acid properties and other features and (iii) algorithms for discriminating different types of membrane proteins and annotating them in genomic sequences. In addition, we provide a list of online resources for the databases and web servers for functional annotation of membrane proteins.

Spectroscopic Study of Anchoring Aromatic Residues in Membrane Proteins and Peptides: Applications to Protein Folding and Vesicle Disruption

Aromatic amino acids play critical roles in the stability and function of membrane-associated proteins and peptides. Here, we apply the site-selective vibrational tool, UV resonance Raman spectroscopy, to probe changes in the structure and microenvironment of tryptophan residues in integral membrane proteins and membrane-associated antimicrobial peptides. Alterations in molecular interactions, such as hydrogen-bonding states, cation-pi interactions, and local polarity, of tryptophan residues accompany the association and folding of these membrane-bound proteins to synthetic lipid bilayers. These results reveal the diversity of molecular interactions that help guide the in vitro assembly of membrane proteins and peptides in vesicles, and provide molecular clues to the mechanisms of membrane protein folding and vesicle disruption.

Hydrogen Bonding and Solvent Polarity Markers in the UV Resonance Raman Spectrum of Tryptophan: Application to Membrane Proteins

Ultraviolet resonance Raman (UVRR) spectra of tryptophan compounds in various solvents and a model peptide are presented and reveal systematic changes that reflect solvent polarity, hydrogen bond strength, and cation-pi interaction. The commonly utilized UVRR spectral marker for environment polarity that has been based on off-resonance Raman data, the tryptophan Fermi doublet ratio I1360/I1340, exhibits different values in on- and off-resonance Raman spectra as well as for different tryptophan derivatives. Specifically, the UVRR Fermi doublet ratio for indole ranges from 0.3 in polar solvents to 0.8 in nonpolar solvents, whereas the respective values reported here and previously for off-resonance Raman spectra are 0.5-1.3. UVRR Fermi doublet ratios for the more biologically relevant molecule, N-acetyl tryptophan ethyl ester (NATEE), are in a smaller range of 1.1 (polar solvent) to 1.7 (nonpolar solvent) and correlate to the solvent polarity/polarization parameters pi* and ETN. As has been reported previously, several UVRR modes are also sensitive to the hydrogen bond strength of the indole N-H moiety. Here, we report a new unambiguous marker for H-bonding: the ratio of the W10 (approximately 1237 cm-1) intensity to that of the W9 (approximately 1254 cm-1) mode (RW10). This ratio is 0.7 for NATEE in the absence of hydrogen bond acceptors and increases to 3.1 in the presence of strong hydrogen bond acceptors, with a value of 2.3 in water. The W8 and W17 modes shift more than +10 and approximately -5 cm-1 upon increase in hydrogen bond strength; this range for W17 is smaller than that reported previously and reflects a more realistic range for proteins and peptides in solution. Finally, our data provide evidence for change in the W18 and W16 relative intensity in the presence of cation-pi interactions. These UVRR markers are utilized to interpret spectra of model membrane-bound systems tryptophan octyl ester and the peptide toxin melittin. These spectra reveal the importance of intra- and intermolecular hydrogen bonding and cation-pi interactions that likely influence the partitioning of membrane-associated biomolecules to lipid bilayers or self-associated soluble oligomers. The UVRR analysis presented here modifies and augments prior reports and provides an unambiguous set of spectral makers that can be applied to elucidate the molecular microenvironment and structure of a wide range of complex systems, including anchoring tryptophan residues in membrane proteins and peptides.

Comparative analysis of the packing topology of structurally important residues in helical membrane and soluble proteins

Elucidating the distinct topology of residue packing in transmembrane proteins is essential for developing high-quality computational tools for their structure prediction. Network approaches transforming a protein's three-dimensional structure into a network have proven useful in analyzing various aspects of protein structures. Residues with high degree of connectivity as identified through network analysis are considered to be important for the stability of a protein's folded structure. It is thus of interest to study the packing topology of these structurally important residues in membrane proteins. In this work, we systematically characterized the importance and the spatial topology of these highly connected residues in helical membrane and helical soluble proteins from several aspects. A representative helical membrane protein and two helical soluble protein structure data sets were compiled and analyzed. Results of analyses indicate that the highly connected amino acid residues in membrane proteins are more scattered peripherally and more exposed to the membrane than in soluble proteins. Accordingly, they are less densely connected with each other in membrane proteins than in soluble proteins. Together with the knowledge of a centralized function site for many membrane proteins, these findings suggest a structure-function model that is distinguishable from soluble proteins.

TMFunction: database for functional residues in membrane proteins

We have developed the database TMFunction, which is a collection of more than 2900 experimentally observed functional residues in membrane proteins. Each entry includes the numerical values for the parameters IC50 (measure of the effectiveness of a compound in inhibiting biological function), Vmax (maximal velocity of transport), relative activity of mutants with respect to wild-type protein, binding affinity, dissociation constant, etc., which are important for understanding the sequence–structure–function relationship of membrane proteins. In addition, we have provided information about name and source of the protein, Uniprot and Protein Data Bank codes, mutational and literature information. Furthermore, TMFunction is linked to related databases and other resources. We have set up a web interface with different search and display options so that users have the ability to get the data in several ways. TMFunction is freely available at http://tmbeta-genome.cbrc.jp/TMFunction/.

Functional discrimination of membrane proteins using machine learning techniques

BackgroundDiscriminating membrane proteins based on their functions is an important task in genome annotation. In this work, we have analyzed the characteristic features of amino acid residues in membrane proteins that perform major functions, such as channels/pores, electrochemical potential-driven transporters and primary active transporters.ResultsWe observed that the residues Asp, Asn and Tyr are dominant in channels/pores whereas the composition of hydrophobic residues, Phe, Gly, Ile, Leu and Val is high in electrochemical potential-driven transporters. The composition of all the amino acids in primary active transporters lies in between other two classes of proteins. We have utilized different machine learning algorithms, such as, Bayes rule, Logistic function, Neural network, Support vector machine, Decision tree etc. for discriminating these classes of proteins. We observed that most of the algorithms have discriminated them with similar accuracy. The neural network method discriminated the channels/pores, electrochemical potential-driven transporters and active transporters with the 5-fold cross validation accuracy of 64% in a data set of 1718 membrane proteins. The application of amino acid occurrence improved the overall accuracy to 68%. In addition, we have discriminated transporters from other α-helical and β-barrel membrane proteins with the accuracy of 85% using k-nearest neighbor method. The classification of transporters and all other proteins (globular and membrane) showed the accuracy of 82%.ConclusionThe performance of discrimination with amino acid occurrence is better than that with amino acid composition. We suggest that this method could be effectively used to discriminate transporters from all other globular and membrane proteins, and classify them into channels/pores, electrochemical and active transporters.

Is There a Preferential Interaction between Cholesterol and Tryptophan Residues in Membrane Proteins?

Recently, several indications have been found that suggest a preferential interaction between cholesterol and tryptophan residues located near the membrane-water interface. The aim of this study was to investigate by direct methods how tryptophan and cholesterol interact with each other and what the possible consequences are for membrane organization. For this purpose, we used cholesterol-containing model membranes of dimyristoylphosphatidylcholine (DMPC) in which a transmembrane model peptide with flanking tryptophans [acetyl-GWW(LA)8LWWA-amide], called WALP23, was incorporated to mimic interfacial tryptophans of membrane proteins. These model systems were studied with two complementary methods. (1) Steady-state and time-resolved Förster resonance energy transfer (FRET) experiments employing the fluorescent cholesterol analogue dehydroergosterol (DHE) in combination with a competition experiment with cholesterol were used to obtain information about the distribution of cholesterol in the bilayer in the presence of WALP23. The results were consistent with a random distribution of cholesterol which indicates that cholesterol and interfacial tryptophans are not preferentially located next to each other in these bilayer systems. (2) Solid-state 2H NMR experiments employing either deuterated cholesterol or indole ring-deuterated WALP23 peptides were performed to study the orientation and dynamics of both molecules. The results showed that the quadrupolar splittings of labeled cholesterol were not affected by an interaction with tryptophan-flanked peptides and, vice versa, that the quadrupolar splittings of labeled indole rings in WALP23 are not significantly influenced by addition of cholesterol to the bilayer. Therefore, both NMR and fluorescence spectroscopy results independently show that, at least in the model systems studied here, there is no evidence for a preferential interaction between cholesterol and tryptophans located at the bilayer interface.

Co-evolving residues in membrane proteins

The analysis of co-evolving residues has been exhaustively evaluated for the prediction of intramolecular amino acid contacts in soluble proteins. Although a variety of different methods for the detection of these co-evolving residues have been developed, the fraction of correctly predicted contacts remained insufficient for their reliable application in the construction of structural models. Membrane proteins, which constitute between one-fourth and one-third of all proteins in an organism, were only considered in few individual case studies. We present the first general study of correlated mutations in alpha-helical membrane proteins. Using seven different prediction algorithms, we extracted co-evolving residues for 14 membrane proteins having a solved 3D structure. On average, distances between correlated pairs of residues lying on different transmembrane segments were found to be significantly smaller compared to a random prediction. Covariation of residues was frequently found in direct sequence neighborhood to helix-helix contacts. Based on the results obtained from individual prediction methods, we constructed a consensus prediction for every protein in the dataset that combines obtained correlations from different prediction algorithms and simultaneously removes likely false positives. Using this consensus prediction, 53% of all predicted residue pairs were found within one helix turn of an observed helix-helix contact. Based on the combination of co-evolving residues detected with the four best prediction algorithms, interacting helices could be predicted with a specificity of 83% and sensitivity of 42%. http://webclu.bio.wzw.tum.de/helixcorr/

The Role of a Hydrogen Bonding Network in the Transmembrane β-Barrel OMPLA

The hydrogen bonding of polar side-chains has emerged as an important theme for membrane protein interactions. The crystal structure of the dimeric state of the transmembrane β-barrel protein outer membrane phospholipase A (OMPLA) revealed an intermolecular hydrogen bond mediated by a highly conserved glutamine side-chain (Q94). It has been shown that the introduction of a polar residue can drive the association of model helices, and by extension it was presumed that the glutamine hydrogen bond played a key role in stabilizing the OMPLA dimer. However, a thermodynamic investigation using sedimentation equilibrium ultracentrifugation in detergent micelles reveals that the hydrogen bond plays only a very modest role in stabilizing the dimer. The Q94 side-chain is hydrogen bonded intramolecularly to residues Y92 and S96, but amino acid substitutions at these positions suggest these intramolecular interactions are not responsible for attenuating the strength of the intermolecular Q94 hydrogen bond. Other substitutions suggested that hydration of the local environment around Q94 may be responsible for the modest strength of the hydrogen bond. Heat inactivation experiments with the variants suggest that the Y92-Q94-S96 network may instead be important for thermal stability of the monomer. These results highlight the context dependence and broad range of interactions that can be mediated by polar residues in membrane proteins.

Combining Fluorescence Detection and Mass Spectrometric Analysis for Comprehensive and Quantitative Analysis of Redox-Sensitive Cysteines in Native Membrane Proteins

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum Ca2+ release channel is described.

Mapping the proteome of thylakoid membranes byde novo sequencing of intermembrane peptide domains

The proteome of a membrane compartment has been investigated by de novo sequence analysis after tryptic in gel digestion. Protein complexes and corresponding protein subunits were separated by a 2-D Blue Native (BN)/SDS-PAGE system. The transmembrane proteins of thylakoid membranes from a higher plant (Hordeum vulgare L.) were identified by the primary sequence of hydrophilic intermembrane peptide domains using nano ESI-MS/MS-analysis. Peptide analysis revealed that lysine residues of membrane proteins are primarily situated in the intermembrane domains. We concluded that esterification of lysine residues with fluorescent dyes may open the opportunity to label membrane proteins still localized in native protein complexes within the membrane phase. We demonstrate that covalent labelling of membrane proteins with the fluorescent dye Cy3 allows high sensitive visualization of protein complexes after 2-D BN/SDS-PAGE. We show that pre-electrophoretic labelling of protein subunits supplements detection of proteins by post-electrophoretic staining with silver and CBB and assists in completing the identification of the membrane proteome.

Role of Solvent Accessibility in Structure Based Drug Design

Solvent accessibility plays an important role to the structure and function of biological macromolecules. Generally amino acid residues located in the surface of a protein serve as active sites and/or interact with other molecules and ligands. In this review, we briefly define the concept of solvent accessibility with computational procedure. The methods available for calculating solvent accessibility of molecules from their three-dimensional structures and predicting the accessibility from amino acid sequences have been described. The salient features of the solvent accessibility of amino acid residues/nucleotides/sugars in biomolecular structures and its importance for the stability of biomacromolecules have been explored. Further, the application of solvent accessibility for identifying active site residues, binding sites in DNA binding proteins and functionally important residues in membrane proteins will be discussed. The information about the accessible surface area of the basic units of biomacromolecules will be very helpful for de novo protein and drug design. Keywords: solvent accessibility, drug design, active site, structure, prediction

Mutational analysis of the role of tryptophan residues in an antimicrobial peptide.

Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Leu and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.

Comparison of Helix Interactions in Membrane and Soluble α-Bundle Proteins

Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble α-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with respect to membrane protein structure prediction and membrane protein stability.

Protein methylation as a marker of aspartate damage in glucose-6-phosphate dehydrogenase-deficient erythrocytes: role of oxidative stress.

The 'Mediterranean' variant of glucose-6-phosphate dehydrogenase (G6PD) deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants. Previous work has shown increased formation of altered aspartate residues in membrane proteins during cell ageing and in response to oxidative stress in normal erythrocytes. These abnormal residues are specifically recognized by the repair enzyme L-isoaspartate (d-aspartate) protein O-methyltransferase (PCMT; EC 2.1.1.77). The aim of this work was to study the possible involvement of protein aspartate damage in the mechanism linking the G6PD defect and erythrocyte injury, through oxidative stress. Patients affected by G6PD deficiency (Mediterranean variant) were selected. In situ methylation assays were performed by incubating intact erythrocytes in the presence of methyl-labelled methionine. Altered aspartate residues were detected in membrane proteins by methyl ester quantification. We present here evidence that, in G6PD-deficient erythrocytes, damaged residues are significantly increased in membrane proteins, in parallel with the decay of pyruvate kinase activity, used as a cell age marker. Erythrocytes from patients were subjected to oxidative stress in vitro, by treatment with t-butylhydroperoxide, monitored by a rise in concentration of both methaemoglobin and thiobarbituric acid-reactive substances. L-Isoaspartate residues increased dramatically in G6PD-deficient erythrocytes in response to such treatment, compared with baseline conditions. The increased susceptibility of G6PD-deficient erythrocytes to membrane protein aspartate damage in response to oxidative stress suggests the involvement of protein deamidation/isomerization in the mechanisms of cell injury and haemolysis.