A conformational preference parameter to predict helices in integral membrane proteins.

Assignments were made for helical regions in several integral membrane proteins using an algorithm devised to delineate the transmembrane helices in bacteriorhodopsin (Eur. J. Biochem. 182 (1982) 565-575). A new conformational preference parameter for membrane-buried helices was obtained. The use of this parameter to predict helices in membrane proteins is discussed. When applied to the L and M subunits of Rhodopseudomonas sphaeroides, five helices were predicted, which is consistent with the three-dimensional X-ray crystal structure. Data on signal sequences and amino acid exchanges in membrane proteins are also analysed and discussed

Structural homology of lens crystallins. III. Secondary structure estimation from circular dichroism and prediction from amino acid sequences.

Circular dichroism spectra (196-240 nm) of calf alpha-, beta H-, beta L- and gamma-crystallins were measured and analyzed over the entire wavelength range with five curve-fitting procedures for estimating protein secondary structure. For gamma-crystallin the estimates are in good agreement with the X-ray structure. For all four crystallins the estimates are very similar: 0-9% alpha-helix and 51-68% beta-sheet. This is in accordance with the three-dimensional homology of beta Bp- and gamma 2-crystallin polypeptide chains as postulated from their 30% sequence homology, and suggests that alpha A- and alpha B-crystallin chains may also have a corresponding structure. Secondary structure elements in the four amino acid sequences were predicted using two different comprehensive prediction methods. For gamma 2-crystallin the predictions of beta-sheet are in good agreement with the X-ray structure and with circular dichroism estimates. For beta Bp-crystallin only the C-terminal domain secondary structure predictions are considered satisfactory, which possibly relates to the proposed role of the N-terminal domain in subunit interactions. The combined predictions for alpha A- and alpha B-chains (3% helix, 49% sheet) are in excellent agreement with circular dichroism. Moreover, the good alignment of predicted beta-sheet segments in alpha-crystallin chains with known beta-sheet strands in gamma 2- (and presumably beta Bp-) crystallin strongly supports a similar 4-motif folding pattern in all four calf crystallin chains.

An assessment of protein secondary structure prediction methods based on amino acid sequence.

Five of the several secondary structure prediction methods based on protein amino acid sequence has been computerized, allowing the calculation of joint prediction histograms which have been shown to be superior to any individual prediction. The known structures of about 40 proteins experimentally determined by X-ray crystallography are compared with the predictions resulting from calculated histograms. The accuracy of the predictions for helices is generally much better than for both beta-sheet regions and for turns. The overall agreement between prediction and observation within the amino terminal half of the protein molecules is clearly superior to that for the carboxyl half, suggesting an amino nucleating core. Predictions for smaller proteins and thermally stable proteins are generally good, indicating the sensitivity of the methods to short-range but not long-range interactions. In less than half the cases tested were the predictions useful; there was no way of knowing ahead of time if a favorable prediction would result. Given the lack of dramatic improvement with an increase in data base for the schemes and the generally poor agreement factors, it appears that a perfect predictive algorithm must include a consideration of energy minimization, thermalization, and long-range interactions. Extreme caution is suggested in applying present prediction routines to unknown protein structures.

The secondary structure of staphylococcal enterotoxins A, B and C.

The circular dichroism (CD) of staphylococcal enterotoxins A, B and C was measured. The CD of enterotoxins B and C were almost identical from 250 to 320 nm, but differed from the CD of enterotoxin A. The spectrum of enterotoxin A in this wavelength region contained the same bands with respect to both location and sign, but with significant differences in intensity. The CD spectra of enterotoxins B and C were also much more alike from 190 to 250 nm. Although all three enterotoxins had a major negative extremum at 215--218 nm, its magnitude was equal in enterotoxins B and C, but was substantially decreased in enterotoxin A. The secondary structure of the enterotoxins contained little alpha-helix as analyzed with CD models. A secondary structure of entertoxin B compured from a scheme based on a joint prediction histogram of five separate methods, placed 29 residues in alpha-helices, 71 in beta-pleated sheets, 88 in beta-turns and 55 in aperiodic conformation.

Probing Flp: a new approach to analyze the structure of a DNA recognizing protein by combining the genetic algorithm, mutagenesis and non-canonical DNA target sites.

A topological and functional overview of a DNA recognition protein with unknown structure can be achieved by combining three different, but complementary approaches: modeling by the genetic algorithm, functional analysis of mutated variants, and testing the target DNA using non-canonical oligonucleotides. As an example we choose the Flp protein, a site-specific recombinase from Saccharomyces cerevisiae. We derive the topological outline including the DNA binding cleft, examine DNA binding regions by deletional and mutational analysis, and analyze the DNA binding site using 7-deazaadenine, 7-deazaguanine, inosine and 4-O-methylthymine as probes. The combined data offer a comprehensive sketch of a plausible protein architecture for Flp. The structure is detailed enough to verify the prediction accuracy for different peptide regions from pre-existing data and by new experimental design.

Analysis of sequence-similar pentapeptides in unrelated protein tertiary structures. Strategies for protein folding and a guide for site-directed mutagenesis.

From the most recent Brookhaven Protein Co-ordinate Databank, 229 sequence-identical pentapeptide pairs, each found in two unrelated protein structures, were collected; 9115 such pairs differing in only one residue were also gathered. For both samples the main-chain fold was conserved about 20% of the time, despite the different atomic environments presented by the unrelated protein architectures. An analysis of the substituted residues as well as the composition of the sequence-similar pentapeptides allowed several suggestions regarding protein folding mechanisms. An examination of the most frequently observed residue substitutions and their correlation with structural changes in the oligopeptide pairs yields a possible guide for site-directed mutagenesis experiments, especially when no tertiary structural information is at hand.

An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion.

Fifty-one examples of oligopeptides linking protein domains were extracted from the Brookhaven database of three-dimensional protein structures. In general, the peptides displayed specific characteristics in composition, conformation, hydrogen bonding, flexibility and the like. The entire database was then searched for pentapeptides that would optimize these natural linker properties. The oligopeptides found are suggested as general candidates to link protein molecules or domains through gene fusion.

A sensitive procedure to compare amino acid sequences.

Methods are discussed that provide sensitive criteria for detection of weak sequence homologies. They are based on the Dayhoff relatedness odds amino acid exchange matrix and certain residue physical characteristics. The search procedure uses several residue probe lengths in comparing all possible segments of two protein sequences, and search plots are shown with peak values displayed over the entire search length. Alignments are automatically effected using the highest search matrix values and without the necessity of gap penalties. Tests for significance are derived from actual protein sequences rather than a random shuffling procedure.

Analysis of insertions/deletions in protein structures.

An analysis of insertions and deletions (indels) occurring in a databank of multiple sequence alignments based on protein tertiary structure is reported. Indels prefer to be short (1 to 5 residues). The average intervening sequence length between them versus the percentage of residue identity in pairwise alignments shows an exponential behaviour, suggesting a stochastic process such that nearly every loop in an ancestral structure is a possible target for indels during evolution. The results also suggest a limit to the average size of indels accommodated by protein structures. The preferred indel conformations are reverse turn and coil as are the preferred conformations at the indel edges (N- and C-terminal sides). Interruptions in helices and strands were observed as very rare events.

Optimal protocol and trajectory visualization for conformational searches of peptides and proteins.

Conformational searches by molecular dynamics and different types of Monte Carlo or build-up methods usually aim to find the lowest-energy conformation. However, this is often misleading, as the energy functions used in conformational calculations are imprecise. For instance, though positions of local minima defined by the repulsive part of the Lennard-Jones potential are usually altered only slightly by functional modification, the relative depths of the minima could change significantly. Thus, the purpose of conformational searches and, correspondingly, performance criteria should be reformulated and appropriate methods found to extract different local minima from the search trajectory and allow visualization in the search space. Attempts at convergence to the lowest-energy structure should be replaced with efforts to visit a maximum number of different local energy minima with energies within a certain range. We use this quantitative criterion consistently to evaluate performances of different search procedures. To utilize information generated in the course of simulation, a "stack" of low energy conformations is created and stored. It keeps track of variables and visit numbers for the best representatives of different conformational families. To visualize the search, projection of multidimensional walks onto a principal plane defined by a set of reference structures is used. With Met-enkephalin as a structural example and a Monte Carlo procedure combined with energy minimization (MCM) as a basic search method, we analyzed the influence on search efficiency of different characteristics as temperature schedules, the step size for variable modification, constrained random step and response mechanisms to search difficulties. Simulated annealing MCM had comparable efficiency with MCM at constant and elevated temperature (about 600 K). Constraining the randomized choice of side-chain chi angles to optimal values (rotamers) on every MCM step did not improve, but rather worsened, the search efficiency. Two low-energy Met-enkephalin conformations with parallel Tyr1 and Phe4 rings, a gamma-turn around the Gly2 residue, and Phe4 and Met5 side-chains forming together a compact hydrophobic cluster were found and are suggested as possible structural candidates for interaction with a receptor or a membrane.

Prediction of transmembrane segments in proteins utilising multiple sequence alignments.

A method for prediction of transmembrane segments from multiply aligned amino acid sequences is presented. For the calculations, two sets of propensity values were used: one for the middle, hydrophobic portion and one for the terminal regions of the transmembrane sequence spans. Average propensity values were calculated for each position along the alignment, with the contribution from each sequence weighted according to its dissimilarity relative to the other aligned sequences. Eight-residue segments were considered as potential cores of transmembrane segments and elongated if their middle propensity values were above a given threshold. End propensity values were also considered as stop signals. Only helices with length of 15 to 29 residues were allowed and corrections for strictly conserved charged residues were also made. The method is shown to be more successful than predictions based upon single sequences alone. In the test set of 28 families with 126 transmembrane segments, only five spans were not predicted or constituted false positives. The method is applied to sequence families for which data on transmembrane segments do not exist or are sparse or contradictory included voltage-gated potassium-channels, cytochrome c oxidases, NADH-ubiquinone oxidoreductase, beta-glucosides-specific phosphotransferase enzyme and major surface antigen of hepatitis B virus.

Evolution of protein cores. Constraints in point mutations as observed in globin tertiary structures.

The amino acid sequences of ten globin chain tertiary structures were aligned and structurally equivalenced by spatial superposition of main-chain C alpha atoms. A search was then performed for structurally equivalent residue pairs that were buried in the protein core and that had mutated but maintained similar unmutated environments. Residues with atoms in contact with such central residue pairs define their environments. Such examples of point mutations would represent in vivo site-directed mutagenesis as would be observed in evolution. A search for mutated but exposed equivalent central residues was also performed. The constraints placed on the characteristics of the mutated residues (e.g., side-chain volume, polarity, radius of gyration) allow suggestions for the evolutionary modes of protein core and surface development as well as residue substitution guidelines to maintain structural stability in protein engineering and design.

Motif recognition and alignment for many sequences by comparison of dot-matrices.

Calculation of dot-matrices is a widespread tool in the search for sequence similarities. When sequences are distant, even this approach may fail to point out common regions. If several plots calculated for all members of a sequence set consistently displayed a similarity between them, this would increase its credibility. We present an algorithm to delineate dot-plot agreement. A novel procedure based on matrix multiplication is developed to identify common patterns and reliably aligned regions in a set of distantly related sequences. The algorithm finds motifs independent of input sequence lengths and reduces the dependence on gap penalties. When sequences share greater similarity, the same approach converts to a multiple sequence alignment procedure.

Side-chain clusters in protein structures and their role in protein folding.

A method has been developed to detect dense clusters of residue side-chains in proteins, where contact is based upon the percentage of the maximum possible for a given residue type. The clusters represent protein sites with the highest degree of interaction amongst their member residues, while contacts with the environment surrounding the cluster are lower in number. The method has been applied to three distinct structural sets of proteins to check for consistency: mixed alpha-helical/beta-sheet proteins, all beta-strand proteins, and all alpha-helical proteins. A number of cluster features generated from these sets are of general interest for protein folding. (1) A majority of the clusters, comprising three to four residues on average, are localized near the protein surfaces and not within the protein cores. (2) The clusters have preferences for the N- and C-terminal ends of alpha-helices and beta-strands in alpha/beta and alpha-proteins, while beta-proteins utilize the middle strand regions more often. A number of clusters connect three or more beta-strands and/or alpha-helices. (3) More than half of the clusters display residue pairs with oppositely charged atoms within 4.5 A of each other. (4) The residue composition of the clusters does not show correlation with hydrophobicity measures but rather with side-chain volume and surface. The highly preferred cluster residues are (in order of decreasing preference) Trp, His, Arg, Tyr, Glu, Gln and Phe. Clusters with extensive internal contacts in related haemoglobin and immunoglobulin tertiary structures show respective conservation. Several examples illustrate "strategic" folding positions in proteins that often bring together a number of sheets and/or helices, suggesting a folding model in which largely preformed secondary structures are joined together in a cluster induced collapse. Alternatively, the clusters may form at some stage in the folding process to reduce considerably the searchable conformational space and help maintain the proper folding pathway. The clusters also provide hints for site-directed mutagenesis and protein engineering experiments as they are also suggested to be important for structural stability.

Suggestions for "safe" residue substitutions in site-directed mutagenesis.

The conserved topological structure observed in various molecular families such as globins or cytochromes c allows structural equivalencing of residues in every homologous structure and defines in a coherent way a global alignment in each sequence family. A search was performed for equivalent residue pairs in various topological families that were buried in protein cores or exposed at the protein surface and that had mutated but maintained similar unmutated environments. Amino acid residues with atoms in contact with the mutated residue pairs defined the environment. Matrices of preferred amino acid exchanges were then constructed and preferred or avoided amino acid substitutions deduced. Given the conserved atomic neighborhoods, such natural in vivo substitutions are subject to similar constrains as point mutations performed in site-directed mutagenesis experiments. The exchange matrices should provide guidelines for "safe" amino acid substitutions least likely to disturb the protein structure, either locally or in its overall folding pathway, and most likely to allow probing the structural and functional significance of the substituted site.

Recognition of distantly related protein sequences using conserved motifs and neural networks.

A sensitive technique for protein sequence motif recognition based on neural networks has been developed. It involves three major steps. (1) At each appropriate alignment position of a set of N matched sequences, a set of N aligned oligopeptides is specified with preselected window length. N neural nets are subsequently and successively trained on N-1 amino acid spans after eliminating each ith oligopeptide. A test for recognition of each of the ith spans is performed. The average neural net recognition over N such trials is used as a measure of conservation for the particular windowed region of the multiple alignment. This process is repeated for all possible spans of given length in the multiple alignment. (2) The M most conserved regions are regarded as motifs and the oligopeptides within each are used to train intensively M individual neural networks. (3) The M networks are then applied in a search for related primary structures in a databank of known protein sequences. The oligopeptide spans in the database sequence with strongest neural net output for each of the M networks are saved and then scored according to the output signals and the proper combination that follows the expected N- to C-terminal sequence order. The motifs from the database with highest similarity scores can then be used to retrain the M neural nets, which can be subsequently utilized for further searches in the databank, thus providing even greater sensitivity to recognize distant familial proteins. This technique was successfully applied to the integrase, DNA-polymerase and immunoglobulin families.

The role of side-chain hydrogen bonds in the formation and stabilization of secondary structure in soluble proteins.

Intra-molecular side-chain:main-chain (sch:mch) and side-chain (sch:sch) hydrogen bonds observed in 44 well refined crystallographic protein structures with non-homologous sequences have been identified, classified and analysed to detect recurring structural patterns. Each observed bond was characterized by the position of its acceptor and donor groups relative to the N and C termini of the particular secondary structure in which they occur and according to their appearance within the same of sequentially separated secondary structures. The role of short-range hydrogen bonds in the formation and stabilization of a secondary structure and the importance of long-range hydrogen bonds as a cohesive force for different structural segments were also examined. It was found that the N terminus of alpha-helices is characterized by recurring sch:mch and sch:sch bonds with elements of the preceding coil segment, while at the C terminus a frequent intra-helix sch:mch hydrogen bond was frequently observed. The residues at or near the beta-strand termini often cross-linked, through hydrogen-bonding, non-sequential coil segments. Coil structures were characterized by recurring, internal sch:mch hydrogen bonding involving small polar side-chain groups situated at or near their N termini (coil N-capping). The significance of hydrogen bonds as formers and stabilizers of a protein fold and the association of its secondary structural units was also considered through an examination of bond density and distribution throughout the protein tertiary structure.

Folding the main chain of small proteins with the genetic algorithm.

Grid-free protein folding simulations were effected using the genetic algorithm, a backbone representation and standard dihedral angular conformations. The topological folding of idealized four-helix bundles was investigated in detail to differentiate among the important protein folding forces used as fitness criteria. Hydrophobic interactions were the most significant while local forces and hydrogen bonds were far less effective in promoting folding. Stable secondary structural regions were also important as nucleating centers. Using the fitness parameters optimized in idealized simulations together with standard secondary structure predictions derived from the amino acid sequence alone, the proper main-chain folding of the four-helix bundle proteins cytochrome b562, cytochrome c' and hemerythrin was achieved. In addition the backbone topology as predicted by the genetic algorithm for crambin, a mixed helix/strand protein with known structure, is presented and discussed.

Identifying the tertiary fold of small proteins with different topologies from sequence and secondary structure using the genetic algorithm and extended criteria specific for strand regions.

Grid-free protein folding simulations based on sequence and secondary structure knowledge (using mostly experimentally determined secondary structure information but also analysing results from secondary structure predictions) were investigated using the genetic algorithm, a backbone representation, and standard dihedral angular conformations. Optimal structures are selected according to basic protein building principles. Having previously applied this approach to proteins with helical topology, we have now developed additional criteria and weights for beta-strand-containing proteins, validated them on four small beta-strand-rich proteins with different topologies, and tested the general performance of the method on many further examples from known protein structures with mixed secondary structural type and less than 100 amino acid residues. Topology predictions close to the observed experimental structures were obtained in four test cases together with fitness values that correlated with the similarity of the predicted topology to the observed structures. Root-mean-square deviation values of C alpha atoms in the superposed predicted and observed structures, the latter of which had different topologies, were between 4.5 and 5.5 A(2.9 to 5.1 A without loops). Including 15 further protein examples with unique folds, root-mean-square deviation values ranged between 1.8 and 6.9 A with loop regions and averaged 5.3 A and 4.3 A, including and excluding loop regions, respectively.

Protein thermal stability, hydrogen bonds, and ion pairs.

Researchers in both academia and industry have expressed strong interest in comprehending the mechanisms responsible for enhancing the thermostability of proteins. Many and different structural principles have been postulated for the increased stability. Here, 16 families of proteins with different thermal stability were theoretically examined by comparing their respective fractional polar atom surface areas and the number and type of hydrogen bonds and salt links between explicit protein atoms. In over 80% of the families, correlations were found between the thermostability of the familial members and an increase in the number of hydrogen bonds as well as an increase in the fractional polar surface which results in added hydrogen bonding density to water. Thus increased hydrogen bonding may provide the most general explanation for thermal stability in proteins. The number of ion pairs was also found to increase with thermal stability in two-thirds of the families tested; however, their rate of addition was only about one-sixth that for internal hydrogen bonds amongst the protein atoms. The preferred residue exchanges and surface atom types useful in engineering enhanced stability were also examined.