Structural adaptation of enzymes to low temperatures.

A systematic comparative analysis of 21 psychrophilic enzymes belonging to different structural families from prokaryotic and eukaryotic organisms is reported. The sequences of these enzymes were multiply aligned to 427 homologous proteins from mesophiles and thermophiles. The net flux of amino acid exchanges from meso/thermophilic to psychrophilic enzymes was measured. To assign the observed preferred exchanges to different structural environments, such as secondary structure, solvent accessibility and subunit interfaces, homology modeling was utilized to predict the secondary structure and accessibility of amino acid residues for the psychrophilic enzymes for which no experimental three-dimensional structure is available. Our results show a clear tendency for the charged residues Arg and Glu to be replaced at exposed sites on alpha-helices by Lys and Ala, respectively, in the direction from 'hot' to 'cold' enzymes. Val is replaced by Ala at buried regions in alpha-helices. Compositional analysis of psychrophilic enzymes shows a significant increase in Ala and Asn and a decrease in Arg at exposed sites. Buried sites in beta-strands tend to be depleted of VAL: Possible implications of the observed structural variations for protein stability and engineering are discussed.

Strain in protein structures as viewed through nonrotameric side chains: II. effects upon ligand binding.

The relation between the spatial positioning of nonrotameric residues and ligands was studied in 112 tertiary structures of protein-ligand complexes with a crystallographic resolution of

Strain in protein structures as viewed through nonrotameric side chains: I. their position and interaction.

We studied the relative spatial positioning of nonrotameric side chains with atypical and strained dihedral angles in well-refined protein tertiary structures. The analysis was confined to buried protein cores, which are less error prone to side-chain positioning. More than half of the proteins with two or more nonrotameric residues displayed clusters of two or more (and up to five) nonrotameric residues. The clusters exhibited lower average crystallographic temperature factors compared with isolated nonrotameric residues. Nonrotameric clusters showed significantly tighter packing than corresponding rotameric clusters and had distinct residue compositions that did not correlate with amino acid characteristics such as size, hydrophobicity, turn preference, and the like. Such nonrotameric residue biases would suggest that spatially concentrated strain in protein folds would be minimized by lowered vibrational energy. Furthermore, nonrotameric residues avoided helices and strands and mostly preferred coil regions. If they were in the helical conformation, then they preferred to be within N-terminal segments. Proteins 1999;37:30-43.

Recognizing very distant sequence relationships among proteins by family profile analysis.

Family profile analysis (FPA), described in this paper, compares all available homologous amino acid sequences of a target family with the profile of a probe family while conventional sequence profile analysis (Gribskov M, Lüthy R, Eisenberg D. Meth Enzymol 1990;183:146-159) considers only a single target sequence in comparison with the probe family. The increased input of sequence information in FPA expands the range for sequence-based recognition of structural relationships. In the FPA algorithm, Zscores of each of the target sequences, obtained from a probe profile search over all known amino acid sequences, are averaged and then compared with the scores for sequences of 100 reference families in the same probe family search. The resulting F-Zscore of the target family, expressed in "effective standard deviations" of the mean Zscores of the reference families, with value above a threshold of 3.5 indicates a statistically significant evolutionary relationship between the target and probe families. The sensitivity of FPA to sequence information was tested with several protein families where distant relationships have been verified from known tertiary protein architectures, which included vitamin B6-dependent enzymes, (beta/alpha)8-barrel proteins, beta-trefoil proteins, and globins. In comparison to other methods, FPA proved to be significantly more sensitive, finding numerous new homologies. The FPA technique is not only useful to test a suspected relationship between probe and target families but also identifies possible target families in profile searches over all known primary structures.

Reliability of atomic displacement parameters in protein crystal structures.

Mean standard errors in atomic displacement parameters (ADPs) resulting from protein crystal structure determinations are estimated by comparing the ADPs of protein-chain pairs of identical sequence within the same crystal or within different crystals displaying the same or different space groups. The estimated ADP standard errors increase nearly linearly as the resolution decreases - an unexpected result given the nonlinear dependence of the resolution on the amount of diffraction data. The estimated ADP standard errors are larger for side-chain and solvent-exposed atoms than for main-chain and buried atoms and, surprisingly, are also larger for residues in the helical secondary structure relative to other local backbone conformations. The results allow an estimate of the influence of crystallographic refinement restraints on ADP standard errors. Such corrections should be applied when comparing different protein structures.

Easy method to predict solvent accessibility from multiple protein sequence alignments.

An easy and uncomplicated method to predict the solvent accessibility state of a site in a multiple protein sequence alignment is described. The approach is based on amino acid exchange and compositional preference matrices for each of three accessibility states: buried, exposed, and intermediate. Calculations utilized a modified version of the 3D_ali databank, a collection of multiple sequence alignments anchored through protein tertiary structural superpositions. The technique achieves the same accuracy as much more complex methods and thus provides such advantages as computational affordability, facile updating, and easily understood residue substitution patterns useful to biochemists involved in protein engineering, design, and structural prediction. The program is available from the authors; and, due to its simplicity, the algorithm can be readily implemented on any system. For a given alignment site, a hand calculation can yield a comparative prediction.

Automated protein sequence database classification. I. Integration of compositional similarity search, local similarity search, and multiple sequence alignment.

MOTIVATION: Genome sequencing projects require the periodic application of analysis tools that can classify and multiply align related protein sequence domains. Full automation of this task requires an efficient integration of similarity and alignment techniques. RESULTS: We have developed a fully automated process that classifies entire protein sequence databases, resulting in alignment of the homologous sequences. The successive steps of the procedure are based on compositional and local sequence similarity searches followed by multiple sequence alignments. Global similarities are detected from the pairwise comparison of amino acid and dipeptide compositions of each protein. After the elimination of all but one sequence from each detected cluster of closely related proteins, the remaining sequences are compiled in a suffix tree which is self-compared to detect local sequence similarities. Sets of proteins which share similar sequence segments are then weighted according to their closeness and multiply aligned using a fast hierarchical dynamic programming algorithm. Computational strategies were devised to minimize computer processing time and memory space requirements. The accuracy of the sequence classifications has been evaluated for 12 462 primary structures distributed over 341 known families. The percentage of sequences with missed or incorrect family assignments was 6.8% on the test set. This low error level is only twice that of the manually constructed PROSITE database ( 3.4% ) and is substantially better than that found for the automatically built PRODOM database ( 34.9% ). AVAILABILITY: The resulting database, called DOMO, is available through database search routine SRS at Infobiogen (http://www.infobiogen.fr/srs5/), EBI (http://srs.ebi.ac.uk:5000/) and EMBL (http://www.embl-heidelberg.de/srs5/) World Wide Web sites. CONTACT: gracy@infobiogen.fr

Automated protein sequence database classification. II. Delineation Of domain boundaries from sequence similarities.

MOTIVATION: Decomposing each protein into modular domains is a basic prerequisite to classify accurately structural units in biological molecules. Boundaries between domains are indicated by two similar amino acid sequence segments located within the same protein (repeats) or within homologous proteins at notably different distances from their respective N- or C-termini. RESULTS: We have developed an automated method that combines such positional constraints derived from various detected pairwise sequence similarities to delineate the modular organization of proteins. The procedure has been applied to a non-redundant data set of 26 990 proteins whose sequences were taken from the PIR and SWISS-PROT databanks and shared <60% sequence identity amongst pairs. The resultant clustering, delineation and multiple alignment of 24 380 sequence fragments yielded a new database of 4364 domain families. Comparison of the domain collection with that of PRODOM indicates a clear improvement in the number and size of domain families, domain boundaries and multiple sequence alignments. The accuracy and sensitivity of the method are illustrated by results obtained for ankyrin-like repeats and EGF-like modules. AVAILABILITY: The resulting database, called DOMO, is available through the database search routine SRS at Infobiogen (http://www.infobiogen.fr/srs5/), EBI (http://srs.ebi.ac.uk:5000/) and EMBL (http://www.embl-heidelberg.de/srs5/) World Wide Web sites. CONTACT: gracy@infobiogen.fr

Relationships between protein sequence and structure patterns based on residue contacts.

The identification of correlations between sequence patterns and structural motifs is a prerequisite in the development of protein structure prediction methods. The prediction accuracy indicates whether these correlations are discerned. We present an approach to identify long-range relationships between sequence patterns and structural motifs by varying the granulation of the structure description. Since interaction among residues is a major determinant in protein folding, we consider contact environments formed by two triplets of three sequentially neighboring residues and described by vectors whose components express contact strengths on an atomic level. Through testing various classification schemes, including their resolution and optimizing parameters, discernible relationships between sequences and folds are explored. About ten structural contact states, together with information from noncontacting regions, could improve the accuracy of contact prediction.

Accessibility to internal cavities and ligand binding sites monitored by protein crystallographic thermal factors.

Protein structures are flexible both in solution and in the solid state. X-ray crystallographically determined thermal factors monitor the flexibility of protein atoms. A method utilizing such factors is proposed to delineate protein regions through which a ligand can exchange between binding site and bulk solvent. It is based on the assumption that thermally excited protein regions are excellent candidates for opening a ligand channel. Computationally simple and inexpensive, the method analyzes directions from which thermal factors can propagate within the protein, resulting in thermal motion paths (TMPs). Applications to engineered T4 lysozymes, where an artificial internal cavity can host hydrophobic molecules, and to sperm whale myoglobins, where the active site is completely buried, yielded results in agreement with other independent structural observations and with previous hypotheses. Further new features could also be suggested. The proposed TMP analysis could aid molecular dynamics simulation studies as well as time-resolved and site-directed mutagenesis experimental studies, especially given its modest computational expense and its direct roots in experimental results based on thermal factors determined in high-resolution crystallographic studies.

Are knowledge-based potentials derived from protein structure sets discriminative with respect to amino acid types?

The parametric description of residue environments through solvent accessibility, backbone conformation, or pairwise residue-residue distances is the key to the comparison between amino acid types at protein sequence positions and residue locations in structural templates (condition of protein sequence-structure match). For the first time, the research results presented in this study clarify and allow to quantify, on a rigorous statistical basis, to what extent the amino acid type-specific distributions of commonly used environment parameters are discriminative with respect to the 20 amino acid types. Relying on the Bahadur theory, we estimate the probability of error in a single-sequence-structure alignment based on weak or absent discriminative power in a learning database of protein structure. We present the results for many residue environment variables and demonstrate that each fold description parameter is sensitive with respect to only a few amino acid types while indifferent to most of the other amino acid types. Even complex structural characteristics combining solvent-accessible surface area, backbone conformation, and pairwise distances distinguish only some amino acid types, whereas the others remain nondiscriminated. We find that the knowledge-based potentials currently in use treat especially Ala, Asp, Gln, His, Ser, Thr, and Tyr as essentially "average" amino acids. Thus, highly discriminative amino acid types define the alignment register in gapless sequence-structure alignments. The introduction of gaps leads to alignment ambiguities at sequence positions occupied by nondiscriminated amino acid types. Therefore, local sequence-structure alignments produced by techniques with gaps cannot be reliable. Conceptionally new and more sensitive environment parameters must be invented.

Protein-protein crystal-packing contacts.

Protein-protein contacts in monomeric protein crystal structures have been analyzed and compared to the physiological protein-protein contacts in oligomerization. A number of features differentiate the crystal-packing contacts from the natural contacts occurring in multimeric proteins. The area of the protein surface patches involved in packing contacts is generally smaller and its amino acid composition is indistinguishable from that of the protein surface accessible to the solvent. The fraction of protein surface in crystal contacts is very variable and independent of the number of packing contacts. The thermal motion at the crystal packing interface and that of the protein core, even for large packing interfaces, though the tendency is to be closer to that of the core. These results suggest that protein crystallization depends on random protein-protein interactions, which have little in common with physiological protein-protein recognition processes, and that the possibility of engineering macromolecular crystallization to improve crystal quality could be widened.

Applying experimental data to protein fold prediction with the genetic algorithm.

Specific residue interactions as revealed from a few and readily available experiments can be quite important in shaping a protein's tertiary topology by complementing basic and general folding principles. This experimental information is employed in structure prediction (mainchain topology) based on sequence knowledge and the genetic algorithm with its ability to optimize simultaneously many parameters. Examples investigated include the distribution of cysteinyl S-S bonds, protein side-chain ligands to iron-sulfur cages, cofactor-ligands, crosslinks amongst side-chains, and conserved hydrophobic and catalytic residues. Such interactions yield an improvement in the predicted topology (0.4-6.6 A root mean square deviation in the positions of the backbone C alpha-atoms relative to those observed) compared with those resulting from simulations relying only on basic protein folding principles. For several examples the resultant topology depended critically on knowledge of the few and specific interactions such that the relationship between predicted and observed C alpha-positions was near random without their use. The combined methodology (experimental data and the genetic algorithm) should prove helpful in settings where experiment and theory can cooperate in successive steps to elucidate an unknown structure.

Hydrophobic patches on protein subunit interfaces: characteristics and prediction.

Hydrophobic patches, defined as clusters of neighboring apolar atoms deemed accessible on a given protein surface, have been investigated on protein subunit interfaces. The data were taken from known tertiary structures of multimeric protein complexes. Amino acid composition and preference, patch size distribution, and patch contact complementarity across associating subunits were examined and compared with hydrophobic patches found on the solvent-accessible surface of the multimeric complexes. The largest or second largest patch on the accessible surface of the entire subunit was involved in multimeric interfaces in 90% of the cases. These results should prove useful for subunit design and engineering as well as for prediction of subunit interface regions.

Prediction of membrane protein topology utilizing multiple sequence alignments.

A technique for prediction of protein membrane topology (intra- and extracellular sidedness) has been developed. Membrane-spanning segments are first predicted using an algorithm based upon multiply aligned amino acid sequences. The compositional differences in the protein segments exposed at each side of the membrane are then investigated. The ratios are calculated for Asn, Asp, Gly, Phe, Pro, Trp, Tyr, and Val, mostly found on the extracellular side, and for Ala, Arg, Cys, and Lys, mostly occurring on the intracellular side. The consensus over these 12 residue distributions is used for sidedness prediction. The method was developed with a set of 42 protein families for which all but one were correctly predicted with the new algorithm. This represents an improvement over previous techniques. The new method, applied to a set of 12 membrane protein families different from the test set and with recently determined topologies, performed well, with 11 of 12 sidedness assignments agreeing with experimental results. The method has also been applied to several membrane protein families for which the topology has yet to be determined. An electronic prediction service is available at the E-mail address tmap@embl-heidelberg.de and on WWW via http://www.embl-heidelberg.de.

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.

Correlation between side chain mobility and conformation in protein structures.

Thermal factors of protein atoms as determined by X-ray crystallographic techniques show a tendency to be larger in side chains with unfavourable local conformations rather than in those displaying conformational energy minima. It follows that side chain atoms are more mobile if they are in a non-rotameric configuration and that the stereochemistry of protein structures cannot be fully assessed or simulated without consideration of thermal factors that monitor flexibility in various regions of the protein. The observations should also prove useful in protein folding and design.

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.

NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding.

The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2'-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moieties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design.