[Proteomic code]. / Proteomnyi kod.

On the basis of recent fundamentally novel developments in the protein structure a proteomic code is suggested, that would potentially allow to describe sequence, structure, and function of proteins by a spectrum of elementary loop-n-lock units. All major characteristics of the nearly standard units are described, and first five "codons" of the proteomic code are presented with their respective unique sequences, structures, and functions. More such codons are to be discovered, and the general procedure for their identification is described.

Protein folding: looping from hydrophobic nuclei.

Protein structure can be viewed as a compact linear array of nearly standard size closed loops of 25-30 amino acid residues (Berezovsky et al., FEBS Letters 2000; 466: 283-286) irrespective of details of secondary structure. The end-to-end contacts in the loops are likely to be hydrophobic, which is a testable hypothesis. This notion could be verified by direct comparison of the loop maps with Kyte and Doolittle hydropathicity plots. This analysis reveals that most of the ends of the loops are hydrophobic, indeed. The same conclusion is reached on the basis of positional autocorrelation analysis of protein sequences of 23 fully sequenced bacterial genomes. Hydrophobic residues valine, alanine, glycine, leucine, and isoleucine appear preferentially at the 25-30 residues distance one from another. These observations open a new perspective in the understanding of protein structure and folding: a consecutive looping of the polypeptide chain with the loops ending primarily at hydrophobic nuclei.

Distinct stages of protein evolution as suggested by protein sequence analysis.

Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code. The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses. According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first. Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A). It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes. After the fusion of the minigenes, a mosaic of the alphabets would form. Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units. The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25-30 residues (Berezovsky et al., 2000). Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25-30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops. The loop closure stage is followed by the formation of typical folds of 100-200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains. This size was apparently dictated by the optimal ring closure for DNA. In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures. Further gene fusions lead to the formation of modern multidomain proteins. Recombinational gene splicing is likely to have appeared after the DNA circularization stage.

Loop fold nature of globular proteins.

Protein chains make numerous returns in globules, thus forming loops, closed by tight residue-to-residue contacts-closed loops. Previous statistical analysis of the sizes and locations of the closed loops in all major protein folds revealed that the loops have an almost standard contour length of 25-30 amino acid residues and follow one after another along the chain. In this work the closed loops of the major folds are presented in three dimensions. A special image filtering procedure is introduced that allows one to visualize the standard size closed loops for the first time. The loop positions along the sequences are verified by detection of loop-end clusters.

Van der Waals locks: loop-n-lock structure of globular proteins.

In a globular protein the polypeptide chain returns to itself many times, making numerous chain-to-chain contacts. The stability of these contacts is maintained primarily by van der Waals interactions. In this work we isolated and analysed van der Waals contacts that stabilise spatial structures of nine major folds. We suggest a specific way to identify the tightest contacts of prime importance for the stability of a given crystallized protein and introduce the notion of the van der Waals lock. The loops closed by the van der Waals interactions provide a basically novel view of protein globule organization: the loop-n-lock structure. This opens a new perspective in understanding protein folding as well: the consecutive looping of the polypeptide chain and the locking of the loop ends by tight van der Waals interactions.

Protein structure and folding: a new start.

Standard building blocks of proteins--closed loops of 25-30 amino acid residues--have been recently discovered and further characterized by combined efforts of several laboratories. New challenging views on the protein structure, folding, and evolution are introduced by these studies. In particular, the role of van der Waals contacts in protein stability is better understood. They can be considered as locks closing the polypeptide chain returns and forming the loop-n-lock elements. The linearity of the arrangement of the standard loops in the proteins has important evolutionary implications. Selection pressure to maintain the loops of nearly standard size is reflected in the protein sequences as characteristic distance between hydrophobic residues, equal to the loop end-to-end distance. Further characterization of the loop-n-lock units reveals several sequence/structure prototypes, which suggests a new basis for protein classification. The following is a review of these studies.

Hierarchy of regions of amino acid sequence with respect to their role in the protein spatial structure.

The method of the representation of amino acid sequence by graph of the interactions energy between parts of spatial structure has been elaborated. Our method provides the possibility to establish the compatibility between each point of a polypeptide chain and the Van der Waals interactions energy of regions of a native globule adjacent to this amino acid residue. We have undertaken an exhaustive analysis of a set of proteins. Boundaries of domain and module structures have been found. Nonequivalence of different parts of sequences in respect to their contribution to stabilization of the spatial structure of the protein macromolecules has been revealed. On the basis of the number of energetic levels which are necessary to identify all independent parts of the globule, the contribution from each part of the sequence to stabilization of the spatial structure of the globule is defined. Thus, it has been found that the sequence of amino acid residues coincides with the sequence of the numerical values which can be used in turn in formal procedures, such as an alignment, a search of consensus, the recognition of composition peculiarities, etc. An example of the comparison of proteins with various sequence identities is considered to demonstrate the scheme of an alignment procedure.

A new approach for the calculation of the energy of van der Waals interactions in macromolecules of globular proteins.

Van der Waals interaction energy in globular proteins is presented by the interaction energies between regions of protein spatial structure with homogenous medium density distribution. We introduce a notion of the local medium permittivity as a function of absorptance of molecular groups with particular conformation. Proposed theory avoids shortcomings which are typical for the calculations on the basis of the pairwise additive approximation. The approach takes into account local peculiarities of protein spatial structure and physical-chemical characteristics of amino acid residues and molecular groups.

Closed loops of nearly standard size: common basic element of protein structure.

By screening the crystal protein structure database for close Calpha-Calpha contacts, a size distribution of the closed loops is generated. The distribution reveals a maximum at 27+/-5 residues, the same for eukaryotic and prokaryotic proteins. This is apparently a consequence of polymer statistic properties of protein chain trajectory. That is, closure into the loops depends on the flexibility (persistence length) of the chain. The observed preferential loop size is consistent with the theoretical optimal loop closure size. The mapping of the detected unit-size loops on the sequences of major typical folds reveals an almost regular compact consecutive arrangement of the loops. Thus, a novel basic element of protein architecture is discovered; structurally diverse closed loops of the particular size.

Hierarchy of the interaction energy distribution in the spatial structure of globular proteins and the problem of domain definition.

An algorithm for determining of protein domain structure is proposed. Domain structures resulted from the algorithm application have been obtained and compared with available data. The method is based on entirely physical model of van der Waals interactions that reflects as illustrated in this work the distribution of electron density. Various levels of hierarchy in the protein spatial structure are discerned by analysis of the energy interaction between structural units of different scales. Thus the level of energy hierarchy plays role of sole parameter, and the method obviates the use of complicated geometrical criteria with numerous fitting parameters. The algorithm readily and accurately locates domains formed by continuous segments of the protein chain as well as those comprising non-sequential segments, sets no limit to the number of segments in a domain. We have analyzed 309 protein structures. Among 277 structures for which our results could be compared with the domain definitions made in other works, 243 showed complete or partial coincidence, and only in 34 cases the domain structures proved substantially different. The domains delineated with our approach may coincide with reference definition at different levels of the globule hierarchy. Along with defining the domain structure, our approach allows one to consider the protein spatial structure in terms of the spatial distribution of the interaction energy in order to establish the correspondence between the hierarchy of energy distribution and the hierarchy of structural elements.

Amino acid composition of protein termini are biased in different manners.

An exhaustive statistical analysis of the amino acid sequences at the carboxyl (C) and amino (N) termini of proteins and of coding nucleic acid sequences at the 5' side of the stop codons was undertaken. At the N ends, Met and Ala residues are over-represented at the first (+1) position whereas at positions 2 and 5 Thr is preferred. These peculiarities at N-termini are most probably related to the mechanism of initiation of translation (for Met) and to the mechanisms governing the life-span of proteins via regulation of their degradation (for Ala and Thr). We assume that the C-terminal bias facilitates fixation of the C ends on the protein globule by a preference for charged and Cys residues. The terminal biases, a novel feature of protein structure, have to be taken into account when molecular evolution, three-dimensional structure, initiation and termination of translation, protein folding and life-span are concerned. In addition, the bias of protein termini composition is an important feature which should be considered in protein engineering experiments.

Representation of amino acid sequences in terms of interaction energy in protein globules.

We suggest a new simple approach for comparing the primary structure of proteins and their spatial structure. It relies on the one-to-one correspondence between each residue of the polypeptide chain and the energy of van der Waals interactions between the regions of the native globule flanking this residue. The method obviates the sophisticated geometrical criteria for estimating similarity between spatial structures. Besides, it permits one to analyze structural units of different scale.

COOH-terminal decamers in proteins are non-random.

We have undertaken an exhaustive statistical analysis of the amino acid sequences at the carboxyl-terminal (C) ends of proteins. The composition of the C-terminal decapeptides differs from that expected for the given proteins from the overall amino acid composition. For E. coli, yeast, and H. sapiens it was shown that positively charged amino acid residues are over-represented while Gly residues are under-represented. The C-terminal bias, a novel feature of protein structure, should be taken into account when molecular evolution, spatial structure, translational termination and protein folding are concerned.