Sequence divergence analysis for the prediction of seven-helix membrane protein structures: I. Comparison with bacteriorhodopsin

A method using protein sequence divergence to predict the three-dimensionalstructure of the transmembrane domain of seven-helix membraneproteins is described. The key component in the multistep procedureis the calculation of a hydrophilic and lipophilic variabilityindex for each amino acid in an alignment of a family of homologousproteins. The variability profile, a plot of the calculatedvariability index versus alignment position, can be used topredict a tertiary model of the backbone conformation of thetransmembrane domain. This method was applied to bacteriorhodopsin(BR) and the model obtained was compared with the known structureof this protein. Using an alignment of the amino acid sequencesof BR and closely related (20% identity) proteins, the boundariesof the transmembrane regions, their secondary structures andorientations inside the membrane bilayer were predicted basedon the variability profile. Additional information about theshape of the helix bundle was also obtained from the averagevariability of each transmembrane helix with the assumptionthat the helices are packed sequentially and form a closed helixbundle. Correct features of the known structure of BR were foundin the model structure, suggesting that a similar strategy canbe used to predict transmembrane helices and the packing shapeof other membrane proteins with seven transmembrane helices,such as the opsins and other G-protein coupled receptors.     

Helix stability and hydrophobicity in the folding mechanism of the bacterial immunity protein Im9

Recent models suggest that the mechanism of protein folding is determined by the balance between the stability of secondary structural elements and the hydrophobicity of the sequence. Here we determine the role of these factors in the folding kinetics of Im9* by altering the secondary structure propensity or hydrophobicity of helices I, II or IV by the substitution of residues at solvent exposed sites. The folding kinetics of each variant were measured at pH 7.0 and 10 degrees C, under which conditions wild-type Im9* folds with two-state kinetics. We show that increasing the helicity of these sequences in regions known to be structured in the folding intermediate of Im7*, switches the folding of Im9* from a two- to three-state mechanism. By contrast, increasing the hydrophobicity of helices I or IV has no effect on the kinetic folding mechanism. Interestingly, however, increasing the hydrophobicity of solvent-exposed residues in helix II stabilizes the folding intermediate and the rate-limiting transition state, consistent with the view that this helix makes significant non-native interactions during folding. The results highlight the generic importance of intermediates in folding and show that such species can be populated by increasing helical propensity or by stabilizing inter-helix contacts through non-native interactions.     

Relationships between sequence and structure for the four-{alpha}-helix bundle tertiary motif in proteins

The sequences of four--helical bundle proteins are characterizedby a pattern of hydrophilic and hydrophobic amino acids whichis repeated every seven residues. At each position of the heptadrepeat there are specific constraints on the amino acid propertieswhich result from the topology of the tertiary motif. Theseconstraints give rise to patterns of amino acid distributionwhich are distinct from those of other proteins. The distributionsin each of the heptad positions have been determined by a statisticalanalysis of structural and sequence data derived from sevenfamines of aligned protein sequences. The constitution of eachposition is dominated by a very small number of different aminoacids, with the core positions consisting overwhelmingly ofLeu and Ala. The positional preferences of the individual aminoacids can be generally interpreted in terms of residue propertiesand topological constraints. The potential for four-a-helixbundle folding is reflected primarily in the pattern of residueoccurrence in the heptad and not in the overall amino acid compositionof the protein. Possible applications of this analysis in structurepredictions, sequence alignments and in the rational designand engineering of four-a-helkal bundle proteins are discussed.     

Physicochemical factors for discriminating between soluble and membrane proteins: hydrophobicity of helical segments and protein length

The average hydrophobicity of a polypeptide segment is consideredto be the most important factor in the formation of transmembranehelices, and the partitioning of the most hydrophobic (MH) segmentinto the alternative nonpolar environment, a membrane or hydrophobiccore of a globular protein may determine the type of proteinproduced. In order to elucidate the importance of the MH segmentin determining which of the two types of protein results froma given amino acid sequence, we statistically studied the characteristicsof MH helices, longer than 19 residues in length, in 97 membraneproteins whose three-dimensional structure or topology is known,as well as 397 soluble proteins selected from the Protein DataBank. The average hydrophobicity of MH helices in membrane proteinshad a characteristic relationship with the length of the protein.All MH helices in membrane proteins that were longer than 500residues had a hydrophobicity greater than 1.75 (Kyte and Doolittlescale), while the MH helices in membrane proteins smaller than100 residues could be as hydrophilic as 0.1. The possibilityof developing a method to discriminate membrane proteins fromsoluble ones, based on the effect of size on the type of proteinproduced, is discussed.     

Proline residues in transmembrane helices of channel and transport proteins: a molecular modelling study

Proline residues are commonly found in putative transbilayerhelices of many integral membrane proteins which act as transporters,channels and receptors. Intramembranous prolines are often conservedbetween homologous proteins. It has been suggested that suchintrahelical prolines provide liganding sites for cations viaexposure of the backbone carbonyl oxygen atoms of residues i-3and i-4 (relative to the proline). Molecular modelling studieshave been carried out to evaluate this proposal. Bundles ofparallel proline-kinked helices are considered as simplifiedmodels of ion channels. The energetics of K⁺ ion-helix bundleinteractions are explored. It is shown that carbonyl oxygensexposed by the proline-induced kink and at the C-terminus ofthe helices may provide cation-liganding sites. ‘Hybrid’bundles of antiparallel helices, only some of which containproline residues, are considered as models of transport proteins.Again, praline-exposed carbonyi oxygens are shown to be capableof liganding cations. The roles of -helix dipoles and of thegeometry of helix packing are considered in relation to cation-bundleinteractions. Implications with respect to modelling of ionchannel and transport proteins are discussed     

Intrahelical side chain-side chain contacts: the consequences of restricted rotameric states and implications for helix engineering and design

Intrahelical side chain–side chain (sc–sc) interactionsare assumed to play a crucial role in the formation and stabilityof -helices, yet it was found that only 37.2% of all helicalresidues are involved in such close contacts, assuming a specificminimum contact distance. The majority (58.0%) of these weredetected between residues with amino acid sequence spacing i,i + 4. The low frequency of intrahelical sc–sc contactswith sequence separations i, i + 1 and ii, i + 3, each observedwith only about one-third of the i, i + 4 counts, can be directlyand generally attributed to the absence of the g^- conformationin helices for the dihedral angle X₁- However, if it was assumedthat each side chain may maximally make only one sc–sccontact, as most commonly observed, the percentage of contactingpairs increased relative to the maximum possible pairs for agiven sequence spacing by a factor of {small tilde}4, e.g. from20.9 to 81.7% for i, i + 4 contacts. Stereochemical reasonsare also given for the observation that i, i + 3 contacts arecomposed largely of ion or polar pairs, while hydrophobic residuesdominate the i, i + 4 contacts. No significantly increased densityof intrahelical sc–sc contacts with increasing helix lengthwas found. Although there were generally fewer intrahelicalcontacts between buried helical residues when more contactswere made to the tertiary protein environment, the number ofintrahelical contacts did not increase with increasing solventexposure of the helices. Implications for helix design and thepacking of helices are discussed.     

A simple method for predicting transmembrane {alpha} helices with better accuracy

The prediction of a protein's structure from its amino acidsequence has been a long-standing goal of molecular biology.In this work, a new set of conformational parameters for membranespanning helices was developed using the information from thetopology of 70 membrane proteins. Based on these conformationalparameters, a simple algorithm has been formulated to predictthe transmembrane helices in membrane proteins. A FORTRAN programhas been developed which takes the amino acid sequence as inputand gives the predicted transmembrane -helices as output. Thepresent method correctly identifies 295 transmembrane helicalsegments in 70 membrane proteins with only two overpredictions.Furthermore, this method predicts all 45 transmembrane helicesin the photosynthetic reaction center, bacteriorhodopsin andcytochrome c oxidase to an 86% level of accuracy and so is betterthan all other methods published to date.     

Protein sequence entropy is closely related to packing density and hydrophobicity

We investigated the correlation between the Shannon information entropy, 'sequence entropy', with respect to the local flexibility of native globular proteins as described by inverse packing density. These are determined at each residue position for a total set of 130 query proteins, where sequence entropies are calculated from each set of aligned residues. For the accompanying aggregate set of 130 alignments, a strong linear correlation is observed between the calculated sequence entropy and the corresponding inverse packing density determined at an associated residue position. This region of linearity spans the range of C(alpha) packing densities from 12 to 25 amino acids within a sphere of 9 angstrom radius. Three different hydrophobicity scales all mimic the behavior of the sequence entropies. This confirms the idea that the ability to accommodate mutations is strongly dependent on the available space and on the propensity for each amino acid type to be buried. Future applications of these types of methods may prove useful in identifying both core and flexible residues within a protein.     

Maintenance of the hydrophobic face of the diphtheria toxin amphipathic transmembrane helix 1 is essential for the efficient delivery of the catalytic domain to the cytosol of target cells

Abstract The transmembrane (T) domain of diphtheria toxin (DT) comprisesnine -helices and has been shown to play an essential role inthe efficent delivery of the catalytic (C) domain ofDT acrossthe eukaryotic cell membrane and into the cytosol. We have demonstratedrecently thatthe first three amphipathic helixes of the T domain,although not necessary for either channel formation or receptorbinding, are required for the efficient transmembrane deliveryof the Cdomain.In the present study,we have performed a detailedstructure-function analysis of T domainhelix 1 (TH1) of theDT-related fusion protein DAB₃₈₉lL-2. We performed exchangeandsite-directed mutagenesis of TH1 and the resulting mutantfusion toxins were analyzed by gel electrophoresis and testedfor their efficiencies in the delivery of the C domain to thecell cytosol. We demonstrate that the overall charge distributionand hydrophobicity of amino acids in the amphipathic helix TH1,rather than a specific amino acid sequence, are critical forthe function of this helix. The insertion of a charged residuein the hydrophobic face of TH1 abolishes cytotoxic activity,whereas replacement of a hydrophobic residue by a charged aminoacid in the hydrophilic face of the helix has little, if any,effect on cytotoxic activity. In addition,we have identifiedSer220 by site-directed mutagenesis as a residue that appearsto be criticalfor correct folding of the fusion toxin. Mutationsin this position result in fusion proteins that are extremelysensitive to proteolytic attack.     

A holistic approach to protein structure alignment

A method of protein structure comparison developed previouslyis extended to incorporate other aspects of protein structurein addition to the inter-atomic vectors on which it was originallybased. Each additional aspect, which included hydrogen bonding,solvent exposure, torsional angles and sequence, was introducedseparately and evaluated for its ability to improve alignmentquality. The components were then combined, suitably weighted,to produce a more holistic comparison method. The method wastested on a group of remotely related ß/ type proteinsthat share a common feature in their overall chain fold. Theresults indicated that while the original inter-atomic vectorcomponent was sufficient to give the correct alignment of mostpairs of topologically equivalent proteins, the inclusion ofhydrogen bonds, torsion angles and a measure of solvent exposureled to improvements in the more difficult comparisons. Considerationof amino acid properties, including hydrophobicity, had no beneficialeffect. The failure of the latter component was not unexpectedconsidering the almost total lack of sequence similarity amongthe proteins considered.     

Effects of introduced aspartic and glutamic acid residues on the Pi substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated fromSaccharomyces cerevisiae with a preference for Cterminal hydrophobicamino acid residues. In order to alter the inherent substratespecificity of CPD-Y into one for basic amino acid residuesin P'₁, we have introduced Asp and/or Glu residues at a numberof selected positions within the Si binding site. Hie effectsof these substitutions on the substrate specificity, pH dependenceand protein stability have been evaluated. The results presentedhere demonstrate that it is possible to obtain significant changesin the substrate preference by introducing charged amino acidsinto the framework provided by an enzyme with a quite differentspecificity. The introduced acidic amino acid residues providea marked pH dependence of the (k_{cat/Km)FA-A-R-OH/(kcatm})_FA-A-R-OHratio. The change in stability upon introduction of Asp/Gluresidues can be correlated to the difference in the mean buriedsurfac surface area between the substituted and the substitutingamino acid. Thus, the effects of acidic amino acid residueson the protein stability depend upon whether the introducedamino acid protrudes from the solvent accessible surface asdefined by the surrounding residues in the wild type enzymeor is submerged below.     

Effects of amino acid substitutions in the hydrophobic core of {alpha}-lactalbumin on the stability of the molten globule state

Five mutant –lactalbumins, with one or two amino acidsubstitution(s) in the B helix, were engineered to examine therelation between the stability of the molten globule state andthe hydrophobicity of these amino acids. The mutation sites(Thr29, Ala30 and Thr33) have been chosen on the basis of comparisonof the amino acid sequences of goat, bovine and gunea pig –lactalbumin,in which the guinea pig protein shows a remarkably more stablemolten globule than the other proteins. The recombinant proteinswere expressed Escherichia coli and then purified and refoldedefficiently to produce the active proteins. The stability ofthe molten globule state of these engineered proteins has beeninvestigated by urea–induced unfolding transition underan acidic condition (pH 2.0), where the molten globule stateis stable in the absence of urea. The results show that themolten globule state is stabilized by the amino acid substitutionswhich raise the hydrophobicity of the residues, suggesting thatthe hydrophobic core in a globular protein plays an importantrole in the stability of the molten globule state. The changein stabilization free energy of the molten globule state causedby each amino acid substitution has been evaluated, and molecularmechanisms of stabilization of the molten globule state arediscussed.     

Site-specific attachment to recombinant antibodies via introduced surface cysteine residues

Many diagnostic and therapeutic applications of monoclonal antibodiesrequire the covalent linking of effector or reporter moleculesto the immunoglobulin polypeptides. Existing methods generallyinvolve the non-selective modification of amino acid side chains,producing one or more randomly distributed attachment sites.This results in heterogeneous labelling of the antibody moleculesand often to a decrease in antigen-binding due to the modificationof residues close to the antigen-binding site. We report a novelstrategy for site-specifically labelling antibodies throughsurface cysteine residues. Examination of molecular structureswas used to identify amino acids of the C_H1 domain of the IgGheavy chain that were accessible to solvent but not to largermolecules. Site-directed mutagenesis was used to substitutecysteine residues at these positions in the heavy chain of amouse/human chimaeric version of the tumour-binding monoclonalantibody, B72.3. Expression of the modified antibody genes inmammalian cells yielded correctly assembled proteins that hadthiol groups in pre-determined positions and showed no lossof antigen-binding activity. One of the mutants was used todemonstrate the site-specific attachment of a radio-iodinatedligand to the chimaeric B72.3 antibody.     

Predicting surface exposure of amino acids from protein sequence

The amino acid residues on a protein surface play a key rolein interaction with other molecules, determine many physicalproperties, and constrain the structure of the folded protein.A database of monomeric protein crystal structures was usedto teach computer-simulated neural networks rules for predictingsurface exposure from local sequence. These trained networksare able to correctly predict surface exposure for 72% of residuesin a testing set using a binary model (buried/exposed) and for54% of residues using a ternary model (buried/intermediate/exposed).In the ternary model, only 11% of the exposed residues are predictedas buried and only 5% of the buried residues are predicted asexposed. Also, since the networks are able to predict exposurewith a quantitative confidence estimate, it is possible to assignexposure for over half of the residues in a binary model with>80% accuracy. Even more accurate predictions are obtainedby making a consensus prediction of exposure for a homologousfamily. The effect of the local environment of an amino acidon its accessibility, though smaller than expected, is significantand accounts for the higher success rate of prediction thanobtained with previously used criteria. In the absence of athree-dimensional structure, the ability to predict surfaceaccessibility of amino acids directly from the sequence is avaluable tool in choosing sites of chemical modification orspecific mutations and in studies of molecular interaction.     

Comparison of three algorithms for the assignment of secondary structure in proteins: the advantages of a consensus assignment

Accurate assignments of secondary structures in proteins arecrucial for a useful comparison with theoretical predictions.Three major programs which automatically determine the locationof helices and strands are used for this purpose, namely DSSP,P-Curve and Define. Their results have been compared for a non-redundantdatabase of 154 proteins. On a residue per residue basis, thepercentage match score is only 63% between the three methods.While these methods agree on the overall number of residuesin each of the three states (helix, strand or coil), they differon the number of helices or strands, thus implying a wide discrepancyin the length of assigned structural elements. Moreover, thelength distribution of helices and strands points to the existenceof artefacts inherent to each assignment algorithm. To overcomethese difficulties a consensus assignment is proposed whereeach residue is assigned to the state determined by at leasttwo of the three methods. With this assignment the artefactsof each algorithm are attenuated. The residues assigned in thesame state by the three methods are better predicted than theothers. This assignment will thus be useful for analysing thesuccess rate of prediction methods more accurately.     

Understanding the relationship between the primary structure of proteins and their amyloidogenic propensity: clues from inclusion body formation

Amyloid formation is dependent to a considerable extent on the amino acid sequence of the protein. The present study delineates certain sequence-dependent features that are correlated with amyloidogenic propensity. The analyses indicate that amyloid formation is favored by lower thermostability and increased half-life of the protein. There seems to be a certain degree of bias in the composition of order-promoting amino acids in the case of amyloidogenic proteins. Based on these parameters, a prediction function for the amyloidogenic propensity of proteins has been created. The prediction function has been found to rationalize the reported effect of certain mutations on amyloid formation. It seems that a higher sheet propensity of residues that constitute the first seven residues of a helical structure in a protein might increase the propensity for a helix to sheet transition in that region under denaturing conditions.     

Hyperthermostable mutants of Bacillus licheniformis {alpha}-amylase: multiple amino acid replacements and molecular modelling

We have identified previously two critical positions for thethermostability of the highly thermostable -amylase from Bacilluslicheniformis. We have now introduced all 19 possible aminoacid residues to these two positions, His 133 and Ala209. Themost favourable substitutions were to Ile and Val, respectively,which both increased the half-life of the enzyme at 80°Cby a factor of 3. At both positions a stabilizing effect ofhydrophobic residues was observed, although only in the caseof position 133 could a clear correlation be drawn between thehydrophobicity of the inserted amino acid and the gain in proteinstability. The construction of double mutants showed a cumulativeeffect of the most favourable and/or deleterious substitutions.Computer modelling was used to generate a 3-D structure of thewild-type protein and to model substitutions at position 209,which lies in the conserved (/ß)₈ barrel domain of-amylase; Ala209 would be located at the beginning of the thirdhelix of the barrel, in the bottom of a small cavity facingthe fourth helix. The model suggests that replacement by, forexample, a valine could fill this cavity and therefore increaseintra- and interhelical compactness and hydrophobic interactions.     

Computational analysis of alpha-helical membrane protein structure: implications for the prediction of 3D structural models

Relatively little has been known about the structure of alpha-helical membrane proteins, since until recently few structures had been crystallized. These limited data have restricted structural analyses to the prediction of secondary structure, rather than tertiary folds. In order to address this, this paper describes an analysis of the 23 available membrane protein structures. A number of findings are made that are of particular relevance to transmembrane helix packing: (1) on average lipid-tail-accessible transmembrane residues are significantly more hydrophobic, less conserved and contain different residue types to buried residues; (2) charged residues are not always buried and, when accessible to membrane lipid tails, few are paired with another charge and instead they often interact with phospholipid head-groups or with other residue types; (3) a significant proportion of lipid-tail-accessible charged and polar residues form hydrogen bonds only with residues one turn away in the same helix (intra-helix); (4) pore-lining residues are usually hydrophobic and it is difficult to distinguish them from buried residues in terms of either residue type or conservation; and (5) information was gained about the proportion of helices that tend to contribute to lining a pore and the resulting pore diameter. These findings are discussed with relevance to the prediction of membrane protein 3D structure.     

Position-dependent protein mutant profile based on mean force field calculation

The application of the mean force field in protein mutant stabilityprediction is explored. Based on protein main chain characteristics,including polar fraction, accessibility and dihedral angles,the mean force field was constructed to evaluate the compatibilitybetween an amino acid residue and its environment, from whicha position-dependent protein mutant profile was constructed.At each position along a protein sequence, the native residuewas replaced by the other 19 types of amino acid residues. Thematches were evaluated by energies from mean force field calculation,from which a mutant profile along the protein sequence was derived.General characteristics of such a profile were analyzed. Mutantstabilities for two sets of mutants in two proteins were foundto be reasonable compared with experimental data, which indicatesthat the present method can act as a guide in protein engineeringand as an effective scoring matrix in protein sequence–structurealignment studies.