Polarity as a criterion in protein design

Hypothetical proteins can be tested computationally by determiningwhether or not the designed sequence-structure pair has thecharacteristics of a typical globular protein. We have developedsuch a test by deriving quantities with approximately constantvalue for all globular proteins, based on empirical analysisof the exposed and buried surfaces of 128 structurally knownproteins. The characteristic quantities that best appear tosegregate badly designed or deliberately misfolded proteinsfrom their properly folded natural relatives are the polar fractionof side chains on the protein surface and, independently, inthe protein interior. Three of the seven hypothetical structurestested here can be rejected as having too many polar side-chaingroups in the interior or too few on the protein surface. Inaddition, a recently designed nutritional protein is identifiedas being very much unlike globular proteins. These database-derivedcharacteristic quantities are useful in screening designed proteinsprior to experiment and may be useful in screening experimentallydetermined (X-ray, NMR) protein structures for possible errors.     

Designing amino acid sequences to fold with good hydrophobic cores

We present two methods for designing amino acid sequences ofproteins that will fold to have good hydrophobic cores. Giventhe coordinates of the desired target protein or polymer structure,the methods generate sequences of hydrophobic (H) and polar(P) monomers that are intended to fold to these structures.One method designs hydrophobic inside, polar outside; the otherminimizes an energy function in a sequence evolution process.The sequences generated by these methods agree at the levelof 60–80% of the sequence positions in 20 proteins inthe Protein Data Bank. A major challenge in protein design isto create sequences that can fold uniquely, i.e. to a singleconformation rather than to many. While an earlier lattice-basedsequence evolution method was shown not to design unique folders,our method generates unique folders in lattice model tests.These methods may also be useful in designing other types offoldable polymer not based on amino acids     

Modelling of solvent positions around polar groups in proteins

Previous analysis of the distribution of experimental solventmolecule positions around amino acid side chains showed thatdistinct clustering occurred close to polar or charged atomsin proteins. We have used those data to predict likely solventpositions around proteins not used in our initial analysis.We envisage that this algorithm, AQUARIUS, will be useful forfinding solvent positions in electron density maps generatedby protein crystallography and as useful starting positionsfor solvent molecules in computer simulation studies of macromolecules.     

Recognizing misfolded and distorted protein structures by the assumption-based similarity score

A new similarity score (-score) is proposed which is able tofind the correct protein structure among the very close alternativesand to distinguish between correct and deliberately misfoldedstructures. This score is based on the general principle `similarlikes similar', and it favors hydrophobic and hydrophilic contacts,and disfavors hydrophobic-to-hydrophilic contacts in proteins.The values of -scores calculated for the high-resolution proteinstructures from the representative set are compared with thoseof alternatives: (i) very close alternatives which are onlyslightly distorted by conformational energy minimization invacuo; (ii) alternatives with subsequently growing distortions,generated by molecular dynamics simulations in vacuo; (iii)structures derived by molecular dynamics simulation in solventat 300 K; (iv) deliberately misfolded protein models. In nearlyall tested cases the similarity score can successfully distinguishbetween experimental structure and its alternatives, even ifthe root mean square displacement of all heavy atoms is lessthan 1 Å. The confidence interval of the similarity scorewas estimated using the high-resolution X-ray structures ofdomain pairs related by non-crystallographic symmetry. The similarityscore can be used for the evaluation of the general qualityof the protein models, choosing the correct structures amongthe very close alternatives, characterization of models simulatingfolding/unfolding, etc.     

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.     

Alignment of protein sequences using secondary structure: a modified dynamic programming method

A method for comparison of protein sequences based on theirprimary and secondary structure is described. Protein sequencesare annotated with predicted secondary structures (using a modifiedChou and Fasman method). Two lettered code sequences are generated(Xx, where X is the amino acid and x is its annotated secondarystructure). Sequences are compared with a dynamic programmingmethod (STRALIGN) that includes a similarity matrix for boththe amino acids and secondary structures. The similarity valuefor each paired two-lettered code is a linear combination ofsimilarity values for the paired amino acids and their annotatedsecondary structures. The method has been applied to eight globinproteins (28 pairs) for which the X-ray structure is known.For protein pairs with high primary sequence similarity (>45%),STRALIGN alignment is identical to that obtained by a dynamicprogramming method using only primary sequence information.However, alignment of protein pairs with lower primary sequencesimilarity improves significantly with the addition of secondarystructure annotation. Alignment of the pair with the least primarysequence similarity of 16% was improved from 0 to 37% ‘correct’alignment using this method. In addition, STRALIGN was successfullyapplied to seven pairs of distantly related cytochrome c proteins,and three pairs of distantly related picornavirus proteins.     

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.     

Structural identity between the iron- and manganese-containing superoxide dismutases

We have recently reported the first complete amino acid sequenceof an iron-containing superoxide dismutase. The iron enzymeis thought to be closely homologous to the manganese-containingsuperoxide dismutases. The availability of complete amino acidsequence information for four manganese superoxide dismutasesand the crystal structures for two iron and two manganese superoxidedismutases prompted us to investigate the degree of homologybetween the two proteins at various levels. We report that itis not possible to clearly distinguish the two proteins on thebasis of their secondary or tertiary structures. It would appearthat a small number of single site substitutions are responsiblefor conferring distinguishing properties between the two proteins.Substitution of glyclne 77 and glutamine 154 by a glutamineand an alanine respectively in Photobacterium leiognathi ironsuperoxide dismutase may distinguish the kinetic and other particularproperties of this protein from the manganese protein (and otheriron superoxide dismutases). Furthermore the primary structureof both the iron and manganese proteins does not appear to haveany homology with any other known amino acid sequence.     

Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction

De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction.     

A simple approach for protein structure discrimination based on the network pattern of conserved hydrophobic residues

Evolutionarily conserved hydrophobic residues at the core of protein structures are generally assumed to play a structural role in protein folding and stability. Recent studies have implicated that their importance to protein structures is uneven, with a few of them being crucial and the rest of them being secondary. In this work, we explored the possibility of employing this feature of native structures for discriminating non-native structures from native ones. First, we developed a network tool to quantitatively measure the structural contributions of individual amino acid residues. We systematically applied this method to diverse fold-type sets of native proteins. It was confirmed that this method could grasp the essential structural features of native proteins. Next, we applied it to a number of decoy sets of proteins. The results indicate that such an approach indeed identified non-native structures in most test cases. This finding should be of help for the investigation of the fundamental problem of protein structure prediction.     

Conservation of residue interactions in a family of Ca-binding proteins

In the TNC family of Ca-binding proteins (calmodulin, parvalbumin,intestinal calcium binding protein and troponin C) {small tilde}70 well-conserved amino acid sequences and six crystal structuresare known. We find a clear correlation between residue contactsin the structures and residue conservation in the sequences:residues with strong sidechain–sidechain contacts in thethree-dimensional structure tend to be the more conserved inthe sequence. This is one way to quantify the intuitive notionof the importance of sidechain interactions for maintainingprotein three-dimensional structure in evolution and may usefullybe taken into account in planning point mutations in proteinengineering.     

A structure-based model for the halphilic adaptiation of dihydrofolate reductase from Halobacterium volcanii

‘Halophilic adaptation’ of proteins, i.e. the requirementfor high concentrations of monovalent ions for thermodynamicstability of proteins from halophilic organisms, is not fullyunderstood. In this work, an explanation for the halophilicbehavior of dihydrofolate reductase (h-DHFR) from Halobacteriumvolcanii is attempted, based on a model structure derived fromcomparative modeling to dihydrofolate reductase from Escherichiacoli. The model structure of h-DHFR shows an unique asymmetricalcharge distribution over the protein surface, with positivelycharged amino acids centered around the active site and negativecharges on the opposite side of the enzyme. This particularcharge distribution and the correlated molecular dipole arefunctionally relevant. The negative charges on the surface formclusters which are shielded at high salt concentrations; atlow salt, they repulse each other, thus destabilizing the protein.Results are in accordance with denaturation data and, thus,provide an explanation for the exceptional stability propertiesof h-DHFR.     

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.     

A multivariate analysis method for discriminating protein secondary structural segments

Using discriminant analysis, three types of protein secondarystructure segments—helices, ß-strands and coils—arediscriminated by amino acid sequence information alone. A variablein the discriminant analysis is defined by the amino acid indexused to represent the sequence data and by the calculation methodused to extract a feature in this representation. Thus, thethree types of secondary structure segments derived from a setof non-homologous proteins from the Protein Data Bank are analyzedby 888 variables, which correspond to the mean, standard deviation,3.6-residue periodicity and 2-residue periodicity for the numericalprofiles determined from 222 published amino acid indices. Thesevariables are combined to obtain best discrimination of thethree types of segments. When up to three variables are combined,the best discrimination rate was 75%. The variables selectedconsist of the mean of propensity (or turn propensity), themean of ß propensity, and the 3.6-residue periodicityof hydrophobicity. This variable selection procedure can alsobe applied to other types of discrimination problem, once groupsof sequence data are properly organized.     

Solvent interactions with n ring systems in proteins

The interaction of water molecules with apolar amino acids isan important aspect of the hydrophobic effect and hence of proteinfolding. Our distributed multipole electrostatic model for waterinteracting with phenylalanine dipeptides shows that minimumenergy sites exist above the aromatic ring such that a solventmolecule can interact with the electrons, but only when thissite is not blocked by mainchain atoms or disturbed by main-chainpolar atoms. This is consistent with the experimental evidenceof others that water can hydrogen bond to aromatic n electrons.In contrast, our analysis of solvent interactions with phenylalanineresidues based on 48 high-resolution, well-refined protein structuresshows that the dominant interaction of solvent molecules iswith the edge of the ring and not with the 7i electrons. Asthe faces of phenylalanine rings tend to be buried, and solventinteractions with neighbouring polar atoms are more favourable,the interaction of water molecules with the faces of aromatic rings appears not to occur frequently in proteins     

Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.     

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.     

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.     

Local electrostatic optimization in proteins

A simple electrostatic model has been used to investigate theextent to which the structure of protein molecules is organizedto optimize the internal electrostatic interactions. We findthat the model provides a favorable total intra-protein electrostaticenergy for almost all polar and charged groups of atoms, suggestinga high degree of structural optimization. By contrast, a significantfraction of individual group–group interactions are foundto be unfavorable. An analysis as a function of the range ofinteractions included shows the electrostatic organization isgenerally relatively short range (up to 6 or 7 Å betweengroup centers). Although the model is very simple, it is usefulfor assessing the overall quality of protein experimental structures,for pin-pointing some types of errors and as a guide to improvingprotein design.     

An investigation of protein subunit and domain interfaces

Protein structures were collected from the Brookhaven Databaseof tertiary architectures that displayed oligomeric association(24 molecules) or whose polypeptide folding revealed domains(34 proteins). The subunit and domain interfaces for these proteinswere respectively examined from the following aspects: percentagewater-accessible surface area buried by the respective associations,surface compositions and physical characteristics of the residuesinvolved in the subunit and domain contacts, secondary structuralstate of the interface amino acids, preferred polar and non-polarinteractions, spatial distribution of polar and non-polar residueson the interface surface, same residue interactions in the oligomeric:contacts, and overall cross-section and shape of the contactsurfaces. A general, consistent picture emerged for both thedomain and subunit interfaces.