Nature of amino acid substitutions in homologous proteins during evolution

Replacement substitutions of mitochondrial cytochrome $\text{c}$ and α- and β-chains of haemoglobin have been studied by considering the structural similarity among amino acid residues at the secondary and tertiary structural levels. Secondary structural similarity explains ～70% while tertiary structural similarity explains ～50% of observed replacements for most of the cases. These structural similarities could not account for all the replacement substitutions. The study was extended to consider the composition of codons, and the chemical nature and polarity of the replacing and replaced residues. These also could not individually account for all the affected replacements. In general, no property of amino acid residues is conserved for substitutions occurring at any single position during evolution of proteins.     

Distance-constraint approach to higher-order structures of globular proteins with empirically determined distances between amino acid residues

An analysis of higher-order structures of globular proteins by means of a distance-constraint approach is presented. Conformations are generated for each of 21 test proteins of small and medium sizes by optimizing an objective functionf=w _ij(d _ij–d _ij)², whered _ij is a distance between residuesi andj in a calculated conformation, d _ij is an assigned distance to the (ij) pair of residues which is determined based on the statistics of known three-dimensional structures of 14 proteins in the earlier study, andw _ij is a weighting factor. d _ij involves information about hydrophobicity and hydrophilicity of each amino acid residue and about connectivity of a polypeptide chain. In these calculations, only the amino acid sequence is used as input data specific to a calculated protein. With respect to higher-order structures regenerated in the optimized conformations, the following properties are analyzed: (a) N14 of a residue, defined as the number of residues surrounding the residue located within a sphere of radius of 14 Å; (b) root-mean-square differences of the global and local conformations from the corresponding X-ray conformations; (c) distance profiles in the short and medium ranges; and (d) distance maps. The effects of supplementary information about locations of secondary structures and disulfide bonds are also examined to discuss the potential ability of this methodology to predict the three-dimensional structures of globular proteins.     

Automated motif extraction and classification in RNA tertiary structures

We used a novel graph-based approach to extract RNA tertiary motifs. We cataloged them all and clustered them using an innovative graph similarity measure. We applied our method to three widely studied structures: Haloarcula marismortui 50S (H.m 50S), Escherichia coli 50S (E. coli 50S), and Thermus thermophilus 16S (T.th 16S) RNAs. We identified 10 known motifs without any prior knowledge of their shapes or positions. We additionally identified four putative new motifs.     

Annotation of tertiary interactions in RNA structures reveals variations and correlations

RNA tertiary motifs play an important role in RNA folding and biochemical functions. To help interpret the complex organization of RNA tertiary interactions, we comprehensively analyze a data set of 54 high-resolution RNA crystal structures for motif occurrence and correlations. Specifically, we search seven recognized categories of RNA tertiary motifs (coaxial helix, A-minor, ribose zipper, pseudoknot, kissing hairpin, tRNA D-loop/T-loop, and tetraloop-tetraloop receptor) by various computer programs. For the nonredundant RNA data set, we find 613 RNA tertiary interactions, most of which occur in the 16S and 23S rRNAs. An analysis of these motifs reveals the diversity and variety of A-minor motif interactions and the various possible loop-loop receptor interactions that expand upon the tetraloop-tetraloop receptor. Correlations between motifs, such as pseudoknot or coaxial helix with A-minor, reveal higher-order patterns. These findings may ultimately help define tertiary structure restraints for RNA tertiary structure prediction. A complete annotation of the RNA diagrams for our data set is available at http://www.biomath.nyu.edu/motifs/.     

A comparison of fluorescamine and o-phthaldialdehyde as effective blocking reagents in protein sequence analyses by the Beckman sequencer

Use of o-phthaldialdehyde to chemically reduce the newly generated amino termini responsible for the progressively increasing background during an extended amino acid sequence analysis in a liquid phase sequencer has been described. The results have been compared with Fluram blocking using apomyoglobin and rabbit C-reactive protein as standard and unknown samples, respectively.     

Investigation of differences in the tertiary structures of food proteins by small-angle X-ray scattering

Summary With current emphasis in bioengineering on developing new and better structure-function relationships for proteins (e.g., the need for predictability of expected properties prior to cloning), practical and reliable methodology for providing characterization of appropriate features has become of increasing importance. The most potent and detailed technique, X-ray crystallography, has severe limitations: it is so demanding and time-consuming that X-ray coordinates are frequently unavailable for materials of interest; its data relate to static and essentially unhydrated structures, whereas proteins exhibit a variety of dynamic features and function in an aqueous environment; and many proteins of technological importance may never be crystallized. Small-angle X-ray scattering, however, is particularly suitable as a methodology that can provide a substantial number of significant geometric parameters consistent with crystallographic results, that can readily show tertiary structural changes occurring under varying conditions, and that can deal with solutions and gels. Results are presented here from small-angle X-ray scattering investigations of the apo and holo forms of chicken egg-white riboflavin-binding protein, chicken egg-white lysozyme, bovine milk-whey -lactalbumin and -lactoglobulin, and bovine ribonuclease. We utilize these observations to compare tertiary structures of these proteins as well as conformational changes in these structures, and to provide a basis for discussion of their physical and biological significance.Agricultural Research Service, U.S. Department of Agriculture. Reference to brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Methods of locating antigenic determinants of proteins with known primary structures

Theoretical and experimental methods for locating antigenic determinants of proteins with known amino acid sequences are discussed. These methods are systematized on the basis of the theoretical approaches applied, and the efficiency of various predictive methods is compared. Some examples of experimental epitope determination for a number of proteins are given.     

Database of homology-derived protein structures and the structural meaning of sequence alignment

The database of known protein three-dimensional structures can be significantly increased by the use of sequence homology, based on the following observations. (1) The database of known sequences, currently at more than 12,000 proteins, is two orders of magnitude larger than the database of known structures. (2) The currently most powerful method of predicting protein structures is model building by homology. (3) Structural homology can be inferred from the level of sequence similarity. (4) The threshold of sequence similarity sufficient for structural homology depends strongly on the length of the alignment. Here, we first quantify the relation between sequence similarity, structure similarity, and alignment length by an exhaustive survey of alignments between proteins of known structure and report a homology threshold curve as a function of alignment length. We then produce a database of homology-derived secondary structure of proteins (HSSP) by aligning to each protein of known structure all sequences deemed homologous on the basis of the threshold curve. For each known protein structure, the derived database contains the aligned sequences, secondary structure, sequence variability, and sequence profile. Tertiary structures of the aligned sequences are implied, but not modeled explicitly. The database effectively increases the number of known protein structures by a factor of five to more than 1800. The results may be useful in assessing the structural significance of matches in sequence database searches, in deriving preferences and patterns for structure prediction, in elucidating the structural role of conserved residues, and in modeling three-dimensional detail by homology.     

Similarity between average distance maps of structurally homologous proteins

A similarity between average distance maps (Kikuchiet al., 1988a)—that is, predicted contact maps of two tertiary structurally homologous proteins—is examined. Comparisons of shapes of average distance maps (we refer to this as ADM) are made by superpositions of ADMs for two homologous proteins. Also, we compare shapes of actual contact maps for the pair of proteins. We search a optimal superposition mode of each pair of maps showing that two proteins are most similar. It is concluded that two ADMs are also similar when actual tertiary structures between two proteins show similarity. A criterion for similarity of maps is also proposed. The possibility of application of this method to detect weak homology between protein structures is discussed.     

蛋白质序列复杂性简化与非比对序列分析

非比对序列分析是最新发展的一种序列分析方法,具有计算效率高并适用于分析低相似性的序列,已成功用于DNA的序列分析中.但是由于蛋白质序列的复杂性,非比对序列分析对于蛋白质序列分析的准确度却不高.用将20种天然氨基酸残基归类的方法,简化了蛋白质序列的复杂性,并运用到对蛋白质的非比对序列分析中,有效地提高了序列分析的准确性.     

Destabilisation of native tertiary structural interactions is linked to helix-induction by 2,2,2-trifluoroethanol in proteins

The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all β-sheet protein, cardiotoxin analogue 111 (CTX III) from the Taiwan cobra (Naja naja atra) is studied. It is found that high concentrations ( > 80% v/v) of TFE induced a β-sheet to -helix structural transition. It is found that in denatured and reduced CTX III (rCTX III) helical conformation is induced even upon addition of low concentrations ( > 10% v/v) of TFE. Using three other proteins, namely, ribonuclease A (RNase A), lysozyme and -lactalbumin, it is been observed that helix-induction by TFE is intricately linked to drastic destabilization of native tertiary structural interactions in the proteins.     

A critical assessment of comparative molecular modeling of tertiary structures of proteins*

In spite of the tremendous increase in the rate at which protein structures are being determined, there is still an enormous gap between the numbers of known DNA-derived sequences and the numbers of three-dimensional structures. In order to shed light on the biological functions of the molecules, researchers often resort to comparative molecular modeling. Earlier work has shown that when the sequence alignment is in error, then the comparative model is guaranteed to be wrong. In addition, loops, the sites of insertions and deletions in families of homologous proteins, are exceedingly difficult to model. Thus, many of the current problems in comparative molecular modeling are minor versions of the global protein folding problem. In order to assess objectively the current state of comparative molecular modeling, 13 groups submitted blind predictions of seven different proteins of undisclosed tertiary structure. This assessment shows that where sequence identity between the target and the template structure is high (> 70%), comparative molecular modeling is highly successful. On the other hand, automated modeling techniques and sophisticated energy minimization methods fail to improve upon the starting structures when the sequence identity is low (～30%). Based on these results it appears that insertions and deletions are still major problems. Successfully deducing the correct sequence alignment when the local similarity is low is still difficult. We suggest some minimal testing of submitted coordinates that should be required of authors before papers on comparative molecular modeling are accepted for publication in journals. © 1995 Wiley-Liss, Inc.     

Selection of a representative set of structures from Brookhaven Protein Data Bank.

Reliable structural and statistical analyses of three dimensional protein structures should be based on unbiased data. The Protein Data Bank is highly redundant, containing several entries for identical or very similar sequences. A technique was developed for clustering the known structures based on their sequences and contents of alpha- and beta-structures. First, sequences were aligned pairwise. A representative sample of sequences was then obtained by grouping similar sequences together, and selecting a typical representative from each group. The similarity significance threshold needed in the clustering method was found by analyzing similarities of random sequences. Because three dimensional structures for proteins of same structural class are generally more conserved than their sequences, the proteins were clustered also according to their contents of secondary structural elements. The results of these clusterings indicate conservation of alpha- and beta-structures even when sequence similarity is relatively low. An unbiased sample of 103 high resolution structures, representing a wide variety of proteins, was chosen based on the suggestions made by the clustering algorithm. The proteins were divided into structural classes according to their contents and ratios of secondary structural elements. Previous classifications have suffered from subjective view of secondary structures, whereas here the classification was based on backbone geometry. The concise view lead to reclassification of some structures. The representative set of structures facilitates unbiased analyses of relationships between protein sequence, function, and structure as well as of structural characteristics.     

Paired natural cysteine mutation mapping: aid to constraining models of protein tertiary structure.

This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur.     

Secondary structure prediction and unrefined tertiary structure prediction for cyclin A,B, and D

We present heuristic-based predictions of the secondary and tertiary structures of the cyclins A, B, and D, representatives of the cyclin superfamily. The list of suggested constraints for tertiary structure assembly was left unrefined in order to submit this report before an announced crystal structure for cyclin A becomes available. To predict these constraints, a master sequence alignment over 270 positions of cyclin types A, B, and D was adjusted based on individual secondary structure predictions for each type. We used new heuristics for predicting aromatic residues at protein-protein interfaces and to identify sequentially distinct regions in the protein chain that cluster in the folded structure. The boundaries of two conjectured domains in the cyclin fold were predicted based on experimental data in the literature. The domain that is important for interaction of the cyclins with cyclin-dependent kinases (CDKs) is predicted to contain six helices; the second domain in the consensus model contains both helices and a β-sheet that is formed by sequentially distant regions in the protein chain. A plausible phosphorylation site is identified. This work represents a blinded test of the method for prediction of secondary and, to a lesser extent, tertiary structure from a set of homologous protein sequences. Evaluation of our predictions will become possible with the publication of the announced crystal structure.