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.     

Secondary structure prediction of the H5 pore of potassium channels

The ‘H5’ segment located between the putative fifthand sixth transmembrane helices is the most highly conservedregion in voltage-gated potassium channels and it is believedto constitute a major part of the ion conduction path (pore).Here we present a two-step procedure, comprising secondary structureprediction and hydrophobic moment profiling, to predict thestructure of this important region. Combined results from theapplication of the procedure to the H5 region of four voltage-gatedand five other K⁺ channel sequences lead to the prediction ofa ß-strand-turn-(3-strand structure for H5. The reasonsfor the application of these soluble protein methods to partsof membrane proteins are: (i) that pore-lining residues areaccessible to water and (ii) that a large enough database ofhighresolution membrane protein structures does not yet existThe results are compared with experimental results, in particularspectroscopic studies of two peptides based on the H5 sequenceof SHAKER potassium channel. The procedure developed here maybe applicable to wateraccessible regions of other membrane proteins.     

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.     

Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme

To elucidate correlative relationships between structural changeand thermodynamic stability in proteins, a series of mutanthuman lysozymes modified at two buried positions (Ile56 andIle59) were examined. Their thermodynamic parameters of denaturationand crystal structures were studied by calorimetry and X-raycrystallography. The mutants at positions 56 and 59 exhibiteddifferent responses to a series of amino acid substitutions.The changes in stability due to substitutions showed a linearcorrelation with changes in hydrophobicity of substituted residues,having different slopes at each mutation site. However, thestability of each mutant was found to be represented by a uniqueequation involving physical properties calculated from mutantstructures. By fitting present and previous stability data formutant human lysozymes substituted at various positions to theequation, the magnitudes of the hydrophobicity of a carbon atomand the hydrophobicity of nitrogen and neutral oxygen atomswere found to be 0.178 and –0.013 kJ/mol.Ų, respectively.It was also found that the contribution of a hydrogen bond witha length of 3.0 Å to protein stability was 5.1 kJ/moland the entropy loss of newly introduction of a water moleculeswas 7.8 kJ/mol.     

Emulsifying performance of modular beta-sandwich proteins: the hydrophobic moment and conformational stability

Our understanding of protein emulsifying properties is largely based on analysis of emulsifiers found in milk and seed. The 9th-10th type III fibronectin domain pair retains full biological activity following emulsification-encapsulation into polyester microspheres, for controlled delivery, but the conformational criteria determining emulsification efficiency (EE) are unknown. Here, we have generated a series of mutants of this beta-sandwich protein, changing the hydrophobic moment and conformational stability, to investigate the structure-emulsification relationship. Predictive modelling of the hydrophobic moment of beta-strands and mutations known to increase conformational stability were used to generate the series. The proteins were tested for their emulsion stability and EE for oil-in-water mixtures. We show that the stabilization of emulsions by beta-sandwich proteins is best predicted by conformational stability during equilibrium denaturation in ionic surfactant. In contrast, the EE of these proteins is inversely related to an increase in their surface hydrophobicity following unfolding in surfactant. We also describe a novel beta-sandwich emulsifier with strong EE. The requirement for interdomain flexibility to achieve maximum emulsion stability and EE is also shown. This work increases our understanding of the mechanisms involved in protein emulsification and will be of use to the microencapsulation of proteins into polyester microspheres via emulsion-extraction protocols.     

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.     

Protein fragment variability analysis and some principles of protein engineering of vaccines

Based on protein sequence databank (PIR), the ‘variablefragment’ bank, comprising pairs of closely-related proteins,containing one or more strongly differing sites of primary structures,was formed. The bank includes 465 ‘variable fragments’in 383 protein pairs. The amino acid composition of ‘variablefragments’ was examined and indices of potential aminoacid residue variability were formed. An analysis of the interchangeabilityof amino acid fragments depending on the substitution site (N-or C-terminal, or middle part of a chain), the fragment lengthdifferences and physico-chemical properties of residues, suchas volume, hydrophobicity, polarity and isoelectric point, wascarried out. Based on this analysis some general empirical rulesof peptide insertions in carrier proteins were created. Therules are directed at performing modifications leaving the generalstructure and function of the carrier protein molecule unchanged.The selection scheme for the regions suitable for modificationand the criteria for determination of the range of acceptablevariations in these regions were suggested. The use of the potentialvariability profile for detecting regions suitable for peptideinsertion was considered using surface protein of hepatitisB virus as an example.     

The role of extra-membranous inter-helical loops in helix-helix interactions

The effect of a short loop connecting two transmembrane alpha-helices was studied using molecular dynamics simulations. Helices F and G from bacteriorhodopsin and two corresponding polyalanine helices were embedded in octane and POPC membranes in a transmembrane configuration both with and without the inter-helical loop. The results indicate that the membrane environment and the sequence of the loop are more influential on the dynamics and structure of the motif than the presence of a loop as such, at least for the time-scales investigated. The four residues in the FG loop are stabilized by four hydrogen bonds. These hydrogen bonds are not present in the polyalanine loop, causing it to be more flexible than the FG loop. This effect was observed independently of the protein environment, stressing the importance of the sequence. The structural analysis indicates that the loop has weak stabilizing properties in all environments. The stabilization due to the presence of the loop was strongest in a simulation of the FG fragment in a membrane-mimetic octane slab. In the simulations of the helix-loop-helix motif embedded in an explicit lipid bilayer model, the lipid bilayer interface compensates to a large extent for the absence of the loop.     

Spatial sign-alternating charge clusters in globular proteins

Large sign-alternating charge clusters formed by the chargedside groups of amino acid residues and N- and C-terminal groupswere found in the majority of considered globular proteins,namely 235 in a total of 274 protein structures, i.e. 85.8%.The clusters were determined by the criteria proposed earlier:charged groups were included in the cluster if their chargedN and O atoms were located at distances between 2.4 and 7.0Å. The set of selected proteins consisted of known non-homologousprotein structures from the Protein Data Bank with a resolutionless than or equal to 2.5 Å and pair sequence similarityless than 25%. Molecular masses of the proteins were from 5.5to 91.5 kDa and protein chain length from 50 to 830 residues.The distribution of charged groups on the protein surface betweenisolated charged groups, small clusters with two and three groups,and large clusters with four or more groups were found to beapproximately similar making 33, 35 and 32% of the total amountof protein charged groups, respectively. The large sign-alternatingcharge clusters with four or more charged groups were studiedin greater detail. The amount of such clusters depends on theprotein chain length. The small proteins contain 1–3 clusterswhile the large proteins display 4–6 or more clusters.On average, 1.5 clusters per each 100 residues were observed.In contrast with this, the size of a cluster, i.e. the numberof charged groups inside a cluster, does not depend on the proteinmolecular mass, and large clusters are observed for proteinsfrom a range of molecular masses. Clusters consisting of fourto six charged groups occur most frequently, although extralarge clusters are also often revealed. We can conclude thatsign-alternating charge clusters are a common feature of theprotein surface of globular protein. They are suggested to playa general functional role as a local polar factor of proteinsurface.     

Analysis of protein conformational characteristics related to thermostability

The thermal stability of proteins was studied, 195 single aminoacid residue replacements reported elsewhere being analysedfor several protein conformational characteristics: type ofresidue replacement; conservative versus nonconservative substitution;replacement being in a homologous stretch of amino acid residues;change in hydrogen bond, van der Waals and secondary structurepropensities; solvent-accessible versus inaccessible replacement;type of secondary structure involved in the substitution; thephysico-chemical characteristics to which the thermostabilityenhancement can be attributed; and the relationship of the replacementsite to the folding intermediates of the protein, when known.From the above analyses, some general rules arise which suggestwhere amino acid substitutions can be made to enhance proteinthermostability: substitutions are conservative according tothe Dayhoff matrix; mainly occur on conserved stretches of residues;preferentially occur on solvent-accessible residues; maintainor enhance the secondary structure propensity upon substitution;contribute to neutralize the dipole moment of the caps of helicesand strands; and tend to increase the number of potential hydrogenbonding or van der Waals contacts or improve hydrophobic packing.     

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     

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.     

Distance distributions in proteins: a six-parameter representation

We present a statistical analysis of protein structures basedon interatomic C_a distances. The overall distance distributionsreflect in detail the contents of sequence-specific substructuresmaintained by local interactions (such as -helixes) and longerrange interactions (such as disulfide bridges and ß-sheets).We also show that a volume scaling of the distances makes distancedistributions for protein chains of different length superimposable.Distance distributions were also calculated specifically foramino acids separated by a given number of residues. Specificfeatures in these distributions are visible for sequence separationsof up to 20 amino acid residues. A simple representation, whichpreserves most of the information in the distance distributions,was obtained using six parameters only. The parameters giverise to canonical distance intervals and when predicting coarse-graineddistance constraints by methods such as data-driven artificialneural networks, these should preferably be selected from theseintervals. We discuss the use of the six parameters for determiningor reconstructing 3-D protein structures.     

Computer model of a bovine type I collagen microfibril

Collagens are a family of structural proteins of the extracellularmatrix. The fibril-forming collagens are the major structuralproteins of skin, cartilage, bone, blood vessel walls and internalorgans. In addition to biological function, the collagens providenatural structural frameworks that are utilized in the medical,food and leather industries. Many schemes for the organizationof type I collagen into triple helices, microfibrils and fibrilshave been proposed during the past 30 years. Here, the developmentof a molecular model of a bovine type I collagen ‘Smith’microfibril is described. In cross-section, this model exhibitsa symmetrical, pentagonal grouping of five triple helices. Themodel comprises 15 polypeptide chains having 315 residues each.This model is large enough to allow a comparison of its grossstructural features with images of stained collagen obtainedby electron microscopy, yet small enough to be manipulated ona minicomputer or work-station. The model is useful for (amongothers) studies of structure-function relationships in collagen,exploring folding pathways, predicting the efficacy of potentialcrosslinking agents or chemical modifications, and designingsynthetic collagen-like materials or modifications for specificapplications.     

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.     

The prediction and orientation of {alpha}-helices from sequence alignments: the combined use of environment-dependent substitution tables, Fourier transform methods and helix capping rules

Amino acid substitution tables are used to estimate the extentto which amino acids in families of homologous proteins areexposed to the solvent. The approach depends on the comparisonof difference environment-dependent tables for solvent accessible/inaccessibleresidues with amino acid substitutions at each position in analigned set of sequences. The periodicity in the predicted accessible/inaccessibleresidues is calculated using a Fourier transform procedure modifiedfrom that used to calculate hydrophobic moments. a-Helices areidentified from the characteristic periodicities and the solventaccessible face of the helix is defined. The initial helix predictionsare refined using rules for identifying the N- and C-terminiof helices from sequence alignments. These rules have been definedfrom a study of protein structures. The combined method correctlypredicts 79% of the residues in helices and incorrectly predictsonly 12% of the nonhelical residues as helical. In addition,since the method is reliable at predicting the correct numberof helices in the correct position in the sequence and sinceit also predicts the internal face of each helix, the resultscan be used to postulate 3-D arrangements of the secondary structureelements.     

A model for the C5a receptor and for T its interaction with the ligand

A model of the C5a receptor was built based on the assumptionthat the seven membrane-spanning helices of known inward/outwarddirection are in an arrangement roughly similar to that in bacteriorhodopsin.Guidelines for the positioning of the helices were cysteinepairing, ‘ridges into grooves’ interdigitation ofside chains and aromatic cluster formation. The chain segmentsprotruding from the membrane are too short for folding intoan independent ectodomain. The only longer segment (179–202)is tied down in its centre onto the membrane by a disulphidebridge and, thereby, made into two short loops as well. Ideasof the interaction of the C5a receptor with its ligand werederived mainly from the search for accommodation of the functionallyessential arginine residues 40 and 74 of C5a. Asp82 is the onlycharged residue in a pocket {small tilde}20 A below the receptorsurface and is conserved in the rhodopsin superfamily. It commendsitself for binding Arg74 which is the tip of the flexible C-terminalchain of C5a, and rules out Arg40 in the structurally well-definedpart of the molecule. The latter may bind to Glul80 at the bottomof a more shallow pocket which happens to resemble the substrate-bindingsite of trypsin.     

Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.