[The content of alpha-helix and beta-structure in proteins crystallized at different pH values].

Using the data of X-ray diffraction analysis for 100 three-dimensional structures of 26 proteins uniformly distributed among three main classes of the alpha-helix-beta-structure classification and without potentially polyanion regions, 154 comparisons of the content of alpha-helix and beta-structure content were made for structures obtained at different pH values of the medium, being distributed in the whole among all these proteins in the range from 1.5 to 12.0. No significant influence of pH of the medium on the size and localization of alpha-helices and beta-strands was found. As a consequence, it is suggested for the protein structure in a crystal that the alpha-helical-beta-structural backbone of protein structures does not depend on pH of the medium, except when the whole protein or its part can become a polyion so that the electrostatic interactions would either hinder or favour the formation of regular structures, and the conformational properties of ionizable amino acids are independent of pH of the medium. It is unclear, whether these assumptions can be extended to the case of solution, because the data for the structures in solution have been obtained for one protein only. These results can be used in investigations of protein structure, in protein engineering, and in the creation of specialized data banks of protein structures.

[Simulation of alpha-helix and beta-hairpin formation in water-soluble proteins by the code physics method].

One of the possible ways for complete and final solution of the problem of determination of three-dimensional structure of proteins on amino acid sequence is simulation of protein three-dimensional structure formation. The use of the code physics method developed by the author has been suggested to fulfill this task. The simulation of alpha-helix and beta-hairpin formation in water-soluble proteins as a start of realization of the plan is described here. The results of the simulation were compared with the experimental data for 14 proteins of no more than 50 amino acids and therefore with little number of alpha-helices and beta-strands (to meet limits of simulation process) and with secondary structure predictions by the best to data methods of protein secondary structure prediction, PSIpred, PORTER and PROFsec. Secondary structure of the proteins, obtained as a result of the simulation of alpha-helix and beta-hairpin formation using the code physics method, corresponded completely to experimental data while the secondary structure predicted by the PSIpred, PORTER and PROFsec methods differed from these data significantly.

[A comparison of X-ray diffraction analysis and nuclear magnetic resonance from the data on the identification of alpha-helices and beta-strands in the same proteins].

The methods of X-ray diffraction analysis and nuclear magnetic resonance spectroscopy were compared using the data on the identification of alpha-helices and beta-strands of the same proteins. The goal of the study was to determine whether these identifications can be considered as equivalent in the structural classification of proteins and in the solution of other problems. The identifications obtained by the method of Kabsch and Sander for 56% specially chosen water-soluble proteins were chosen. It was found that the identification of alpha-helices and beta-strands are almost equivalent if used for the structural classification of the proteins. In the analysis of the conformational properties of amino acid residues or their combinations, it is reasonable to use the identifications of alpha-helices and beta-strands, obtained only from the data of X-ray diffraction analysis. An additional outcome of the study is a unique collection of pairs of protein structures obtained by the methods of X-ray diffraction analysis and nuclear magnetic resonance spectroscopy for the same proteins.

Amino acid code of protein secondary structure.

The calculation of protein three-dimensional structure from the amino acid sequence is a fundamental problem to be solved. This paper presents principles of the code theory of protein secondary structure, and their consequence--the amino acid code of protein secondary structure. The doublet code model of protein secondary structure, developed earlier by the author (Shestopalov, 1990), is part of this theory. The theory basis are: 1) the name secondary structure is assigned to the conformation, stabilized only by the nearest (intraresidual) and middle-range (at a distance no more than that between residues i and i + 5) interactions; 2) the secondary structure consists of regular (alpha-helical and beta-structural) and irregular (coil) segments; 3) the alpha-helices, beta-strands and coil segments are encoded, respectively, by residue pairs (i, i + 4), (i, i + 2), (i, i = 1), according to the numbers of residues per period, 3.6, 2, 1; 4) all such pairs in the amino acid sequence are codons for elementary structural elements, or structurons; 5) the codons are divided into 21 types depending on their strength, i.e. their encoding capability; 6) overlappings of structurons of one and the same structure generate the longer segments of this structure; 7) overlapping of structurons of different structures is forbidden, and therefore selection of codons is required, the codon selection is hierarchic; 8) the code theory of protein secondary structure generates six variants of the amino acid code of protein secondary structure. There are two possible kinds of model construction based on the theory: the physical one using physical properties of amino acid residues, and the statistical one using results of statistical analysis of a great body of structural data. Some evident consequences of the theory are: a) the theory can be used for calculating the secondary structure from the amino acid sequence as a partial solution of the problem of calculation of protein three-dimensional structure from the amino acid sequence, and the calculated secondary structure and codon strength distribution can be used for simulating the next step of protein folding; b) one can propose that the same secondary structures can be folded into different tertiary structures and, vice versa, different secondary structures can be folded into the same tertiary structures, provided codon distributions are considered also; c) codons can be considered as first elements of protein three-dimensional structure language.

Statistical model of amino acid code of protein secondary structure.

In the previous paper (Shestopalov, 2003) we presented the amino acid code of protein secondary structure as a partial solution of the fundamental problem of the protein three-dimensional structure calculation from the amino acid sequence. Here a statistical model of the code is described. The model is based on the structural data from 2258 protein chains (417,112 amino acid residues used). 60 and 61% of the secondary structure, calculated using the model, coincide, respectively, with the observed secondary structure in the training subset and test subset (104 protein chains and 21,166 residues used). This is equal to the threshold value for all the secondary structure calculations, based on the models, where, similarly as here, only the nearest and middle-range interactions are considered. Therefore the constructed model can be applied for the protein structure prediction from the amino acid sequence, especially when additional information is used along with expert analysis, as in the most successful prediction methods. The model can be used for analysis of the secondary structure changes during protein folding by comparison of the calculated and observed secondary structures. The information about the conformationally invariant segments can serve for the simulation of the supersecondary structure formation. One can try to obtain and examine the protein subset, in which the calculated and observed secondary structures are very similar.

[CHL15--a new gene controlling the replication of chromosomes in saccharomycetes yeast: cloning, physical mapping, sequencing, and sequence analysis]. / CHL15--novyi gen, kontroliruiushchii replikatsiiu khromosom u drozhzhei--sakharomitsetov: klonirovanie, fizicheskoe kartirovanie, sekvenirovanie i funktsional'nyi analiz.

We have analyzed the CHL15 gene, earlier identified in a screen for yeast mutants with increased loss of chromosome III and artificial circular and linear chromosomes in mitosis. Mutations in the CHL15 gene lead to a 100-fold increase in the rate of chromosome III loss per cell division and a 200-fold increase in the rate of marker homozygosis on this chromosome by mitotic recombination. Analysis of segregation of artificial circular minichromosome and artificially generated nonessential marker chromosome fragment indicated that sister chromatid loss (1:0 segregation) is a main reason of chromosome destabilization in the chl15-1 mutant. A genomic clone of CHL15 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL15 revealed a 2.8-kb open reading frame with a 105-kD predicted protein sequence. At the N-terminal region of the protein sequences potentially able to form DNA-binding domains defined as zinc-fingers were found. The C-terminal region of the predicted protein displayed a similarity to sequence of regulatory proteins known as the helix-loop-helix (HLH) proteins. Data on partial deletion analysis suggest that the HLH domain is essential for the function of the CHL15 gene product. Analysis of the upstream untranslated region of CHL15 revealed the presence of the hexamer element, ACGCGT (an MluI restriction site) controlling both the periodic expression and coordinate regulation of the DNA synthesis genes in budding yeast. Deletion in the RAD52 gene, the product of which is involved in double-strand break/recombination repair and replication, leads to a considerable decrease in the growth rate of the chl15 mutant. We suggest that CHL15 is a new DNA synthesis gene in the yeast Saccharomyces cerevisiae.

[Prediction of protein conformation using a doublet code method]. / Predskazanie vtorichnoi struktury belka po metodu dubletnogo koda.

It is suggested that regions of irregular structure, beta-structure, and alpha-helix are composed of 2, 3, and 5 amino acid residue long elements (structurons), respectively, and that the structurons are encoded solely by residue pairs (doublet codons) (i, i + 1), (i, i + 2), (i, i + 4), respectively. Tables of codons are obtained by statistical analysis of the data on the distribution of these pairs in available secondary structures of 62 proteins. These tables are used to obtain distributions of t-, beta- and alpha-codons for an amino acid sequence of protein. When codons of different structures superpose, that is, include the same sequence regions, selection is performed, the selection being performed so to obtain as much as possible number of the non-superposed codons of different structures. The distributions of structurons obtained after this selection are used for localization of structurons in the sequence and prediction of secondary structure on the basis of this localization. The prediction method is illustrated. An accuracy of the method has been tested on the basis an casual selection of fifteen proteins and found equal 64% for secondary structure on the whole and 79%, 53%, 61% for alpha-helix, beta-structure and coil respectively. This result is similar or better than that communicated for contemporary methods.

[DNA-recognizing structure helix-turn-helix in replication initiators from polyomavirus large T antigens]. / DNK-uznaiushchaia struktura spiral'-izgib-spiral' v initsiatorakh replikatsii poliomnykh virusov--bol'shikh T-antigenakh.

Evidence is presented which supports a suggestion that in large T antigens from polyomaviruses-serving as replication initiators--there is DNA-recognizing structure helix-turn-helix, firstly observed in transcription regulators of E. coli and some bacteriophages. It is suggested that all these proteins recognize DNA by the same mechanism.

Amino acid sequence template useful for alpha-helix-turn-alpha-helix prediction.

Necessary stereochemical requirements for an amino acid sequence segment to fold into an alpha-helix-turn-alpha-helix supersecondary structure are presented in sequence template form. The usefulness of the template is illustrated by alpha-helix-turn-alpha-helix predictions consistent with experimental data from the large T antigens of two polyoma viruses, simian virus 40 (segment 143-165) and mouse polyoma virus (segment 297-319), and the yeast transcription activator GCN4 (segment 256-278).

[RNA-recognizing function of the DNA-recognizing structure helix-turn-helix in ribosomal proteins S4 and S7 of Escherichia coli and plant chloroplasts]. / RNK-uznaiushchaia funktsiia DNK-uznaiushchei struktury spiral'-izgib-spiral' v ribosomnykh belkakh S4 i S7 Escherichia coli i khloroplastov rastenii.

The segments which satisfy the necessary requirements for DNA-recognizing structure helix-turn-helix coding have been found in amino acid sequences of RNA-recognizing E. coli ribosomal proteins S4 and S7. It has appeared that among these segments there are those that in chloroplasts S4 and S7 homologues satisfy these necessary requirements also: S4-97-121 and 108-132 (Ec), 91-115 and 102-126 (Zea mays); S7-109-133 (Ec) 110-134 (Euglena gracilis, Glycine max). Such segments have been confronted with known experimental data on 16S rRNA-binding region localization. It has appeared: 1) the segment 97-121 of S4 is localized in RNA-recognizing fragment 46-124 and one of two RNA-binding regions of this fragment containing Lys-120--belongs to the predicted DNA-recognizing structure; the segment 109-133 of S7 includes the only RNA-binding region of S7-113-117. It is concluded that E. coli ribosomal proteins S4 and S7 and their homologues from plant chloroplasts have a DNA-recognizing structure helix-turn-helix performing the RNA-recognizing function.

Structural basis for autogenous regulation of Xenopus laevis ribosomal protein L1 synthesis at the splicing level.

It is known that the injection of the Xenopus laevis ribosomal protein L1 gene into oocytes causes the accumulation of immature L1 transcripts due to a specific block of splicing of the second and third introns. In this paper the secondary structures of these introns in pre-mRNA have been constructed. It has been shown that they share homology with 28 S rRNA. The putative RNA-binding segment of L1 has also been predicted. These results are interpreted as the structural basis for autogenous regulation of X. laevis ribosomal protein L1 synthesis at the splicing level.

[Prediction of the DNA-recognizing supersecondary protein structure, alpha helix-turn-alpha helix, using the modified Ohlendorf-Anderson-Matthews method of necessary stereochemical requirements]. / Predskazatie DNK-uznaiushchei supervtorichnoi struktury belka alpha-spiral'-izgib-alpha-spiral' po metodu modifitsirovannykh neobkhodimykh stereokhimicheskikh uslovii sushchestvovaniia Olendorfa-Andersona-Met'iuza.

A method for prediction of DNA-recognizing supersecondary structure alpha-helix--turn--alpha-helix localization in an amino acid sequence of any protein is described. The method allows to predict this structure in segment 67-89 of E. coli ribosomal protein L7/L12 and in corresponding segments of L7/L12 analogues from other six bacteria and spinach chloroplasts.

[Prediction in histones of the DNA-recognizing protein superstructure alpha helix-turn-alpha helix]. / Predskazanie v gistonakh DNK-uznaiushchei supervtorichnoi struktury belka alpha-spiral'-izgib-alpha-spiral'.

The localization of DNA-recognizing supersecondary structure alpha-helix--turn--alpha-helix in 85 amino acid sequences of histones is predicted. According to the prediction method based on the necessary requirements of amino acid coding this structure may be localized in the following segments of amino acid sequences of calf thymus histones: H1--90--112, H2A--54--76, H2B--50--72 and 102--124, H3--15--37 and 73--95, H4--5--27 or 6--28 and 32--54 or 42--64. According to the known experimental data on the secondary structure of histones only the following localizations are possible: H1--90--112, H2A--54--76, H2B--50--72, H3--73--95, H4--42--64. Using the known experimental data on DNA-histone interactions it is possible to suggest that these localizations of structures alpha-helix--turn--alpha-helix possible in histones H2A, H2B and H4 allows them to participate in close or structurally essential interactions of histones with DNA. The role of the predicted structure in nucleosome formation and in the autoregulation of histone biosynthesis is discussed.

[Secondary structure of histones in solution]. / Issledovanie vtorichnoi struktury gistonov v rastvore.

The secondary structure of histones H2B and H3 from calf thymus has been quantitatively studied in heavy water solutions in a wide range of histone concentrations, pD, and concentrations of sodium chloride by an infrared spectroscopy method. Also, the interactions between molecules of different histones in equimolar mixtures H2A-H2B, H2A-H3, H2A-H4, H2B-H3, H2B-H4, H3-H4, and H2A-H2B-H3-H4 have been investigated using the same method. For H2B and H3 conditions favourable for aggregation have been shown to induce the formation of pleated sheet structure. When the pD and concentration of NaCl are in a physiological range, the secondary structure of H2B and H3 contains about 15% of alpha-helix, 4% of parallel pleated sheet structure, 14% of antipatallel pleated sheet structure in H2B and 18% in H3. For mixtures in all cases, except H2A-H4, there is an interaction between molecules of different histones followed by a reduction of the antiparallel pleated sheet structure content. The data on the secondary structure of histones in different states (under self-association, in mixtures, in nucleosomes, and in chromatin) have been discussed and it is suggested that: 1) the secondary structure of histones in chromatin is essentially similar to that in the state of self-association; 2) in the core nucleosome particle the quantity of DNA (in nucleotide pairs), and the quantities of alpha-helix and antiparallel pleated sheet structure (in peptide groups) satisfy the relation 1 : 1 : 1.

Quantitative study of secondary structure of histones H1, H2A, and H4 in solution by infrared spectroscopy.

The secondary structure of histones H1, H2A, and H4 (F1, F2a2, and F2a1) has been quantitatively studied in heavy water (2H2O) solutions in a wide range of histone concentration, p2H, and concentration of sodium chloride using an improved infrared spectroscopy method. Under all conditions there are about 5--10% of alpha helix. Conditions favourable for aggregation induce formation of antiparallel pleated sheet structure to an extent of about 15% in H1 and H2A and about 30% in H4. When the p2H and concentration of NaCl are in the physiological range, there is the same content of this structure in H2A and H4 and none in H1.