Jinya Otsuka

A comprehensive representation of extensive similarity linkage between large numbers of proteins

A method is described for the representation of a birds-eye view of similarity relationships between large numbers of proteins. With the aid of single-linkage clustering, proteins are clustered into groups on the basis of various types of similarity such as sequence similarity estimated between all the protein pairs. Proteins in a group are directly or indirectly connected to all proteins in the same group by similarities higher than a given threshold and show no similarity higher than the threshold to any proteins outside the group. Thus, all the proteins directly or indirectly related to a protein can be selected out of a large number of proteins by the clustering. Recursion of this clustering of proteins in each group leads to further classification of the proteins. The similarity relationships in each group are visually represented by a similarity matrix. This representation has the advantage of easy detection of the existence of multidomain proteins and diverged families as well as closely related proteins. Such as exhaustive approach to similarity relationships of proteins will be useful for revealing functional/structural/evolutionary units in proteins.

Phylogeny of Organisms Investigated by the Base-Pair Changes in the Stem Regions of Small and Large Ribosomal Subunit RNAs

Abstract. In order to obtain the evolutionary distance data that are as purely additive as possible, we have developed a novel method for evaluating the evolutionary distances from the base-pair changes in stem regions of ribosomal RNAs (rRNAs). The application of this method to small-subunit (SSU) and large-subunit (LSU) rRNAs provides the distance data, with which both the unweighted pair group method of analysis and the neighbor-joining method give almost the same tree topology of most organisms except for some Protoctista, thermophilic bacteria, parasitic organisms, and endosymbionts. Although the evolutionary distances calculated with LSU rRNAs are somewhat longer than those with SSU rRNAs, the difference, probably due to a slight difference in functional constraint, is substantially decreased when the distances are converted into the divergence times of organisms by the measure of the time scale estimated in each type of rRNAs. The divergence times of main branches agree fairly well with the geological record of organisms, at least after the appearance of oxygen-releasing photosynthesis, although the divergence times of Eukaryota, Archaebacteria, and Eubacteria are somewhat overestimated in comparison with the geological record of Earth formation. This result is explained by considering that the mutation rate is determined by the accumulation of misrepairs for DNA damage caused by radiation and that the effect of radiation had been stronger before the oxygen molecules became abundant in the atmosphere of the Earth.

The electronic structure of ferrous heme with imidazole as the fifth ligand. II. Explanation of MCD spectra from the near IR region to the Soret region

On the basis of the ASMO–CI calculation, the MCD associated with x‐ and y‐polarized electronic transitions in the ferrous heme of the high‐spin type is studied by computational and analytical methods. The ground state 5Eη is split into five substates by the mixing of the other low‐lying quintets, 5Eξ and 5B2, through the spin–orbit interaction. The temperature dependent factor in ellipticity is proportional to the z component of orbital angular momentum averaged over the five substates. For excited states, 28 configurations of one‐electron excitations from the three low‐lying quintets are considered; 12 π→π (3a2u, 1a1u→4eg), 14 d→π (u,v→4eg; dπ→2b1u, 3b2u), and 2 d→d (dπ→ū,v) configurations. In the configuration interaction among them, the spin–orbit interactions of the 3d electrons are also taken into account. With the use of the energy eigenvalues and eigenfunctions, MCD curves are computed in the range of wavelength 400–1000 nm. Observed MCD spectra of deoxymyoglobin are well reproduced especial...

Structure model of core proteins in photosystem I inferred from the comparison with those in photosystem II and bacteria; an application of principal component analysis to detect the similar regions between distantly related families of protiens

A principal component analysis based on the physico-chemical properties of amino acid residues is developed to assign similar regions between distantly related families of proteins, taking account of the species diversities in respective families. The most important advantage of this analysis should be that it reflects different physico-chemical properties and thus can predict more detailed structural properties, including the transmembrane helices, than the hydropathy analysis. Its first application reconfirms the similarity between the core proteins of photosynthetic reaction center in purple bacteria and those of photosystem II, indicating that the low percentage of identical amino acid residues estimated previously between them is due to much allowance for amino acid substitutions in purple bacteria. The application of this analysis to the core proteins of photosystem I reveals that any of these proteins includes two domains, each showing high similarity to the amino acid sequences of core proteins in photosystem II and purple bacteria. A core structure model of A1 and A2 proteins folded into four layers of sheets of transmembrane helices is proposed to provide a molecular basis for the electron pathway suggested by spectroscopic experiments as well as for the interaction sites with plastocyanin, 9 kDa protein and LHC proteins.

Discrimination between adaptive and neutral amino acid substitutions in vertebrate hemoglobins

SummaryA discriminant analysis on the basis of the physicochemical properties of amino acid residues is developed to investigate the accumulation pattern of amino acid substitutions in a family of proteins. The application of this analysis to vertebrate hemoglobins reveals the following new results. (1) The major components of teleost fish and amphibian hemoglobins showing the Root effect are sharply discriminated from mammalian hemoglobins in several regions of the α and β chains, whereas shark, minor components of teleost fish and amphibian, reptile, and bird hemoglobins showing no Root effect exhibit a gradual change to mammalian hemoglobin in a straightforward way. This result suggests at least two lines of molecular evolution in vertebrate hemoglobins. (2) The nonadult hemoglobin chains are allocated to the latter line, i.e., tadpole, ξ, and π chains are similar to shark and trout I chains, and ∈ and γ chains are similar to some of the reptile chains. (3) In any case, most of the amino acid residues causing the discrimination are located near the sites that carry the amino acid residues conserved well throughout all classes of vertebrates, suggesting that modifications adapting to the respective living conditions or respiratory organs have taken place effectively near the amino acid residues essential for the manifestation of cooperative oxygen binding. (4) The amino acid residues at other sites are changed from one to another species even within the same class, showing a constant substitution rate as a whole. These amino acid substitutions may be nearly neutral, being under a weak functional constraint. The number of sites allowing such neutral substitutions is rather small, less than one-half of all the sites in the adult hemoglobins of bird and mammal, whereas it amounts to two-thirds in teleost fish hemoglobins.

The electronic structure of ferrous heme with imidazole as the fifth ligand. I. SCF–LCAO–ASMO and CI calculation on the low‐lying multiplets and optically allowed excited states

A semiempirical molecular orbital calculation is performed on the porphin–Fe–imidazole system. As basic atomic orbitals, lone pair σ orbitals of the N atoms coordinating to Fe are employed in addition to 3d ortibals of Fe and π orbitals of N and C atoms. Molecular orbitals are obtained by the usual SCF procedure for the closed shell configuration. Then the calculation of the open shell states is carried out by a careful configuration interaction method. The ground state is identified with one component of 5E. The energy level scheme suggested by magnetic measurements and by ligand field theory for low‐lying quintet, singlet, and triplet states is well reproduced. Corresponding to a broad absorption band in the visible region and weak bands in the near IR, x‐ and y‐polarized electronic transitions, two d→d transitions and six d→π (porphin) transitions as well as π→π transitions, are calculated to be in a range of excitation energy 9–20 kK. Two z‐polarized transitions, d→4dgx (porphin) and d→π′ (imidazole),...

Heitler-London Calculation on the Bonding of Oxygen to Hemoglobin. II. Explanation of the Diamagnetism of Oxyhemoglobin for the Parallel Arrangement of O2 to the Heme

Calculation is carried out for all the triplet and quintet states arising from the low lying terms of the ferrous heme iron atom and those of the oxygen molecule. The charge transfer configurations of ionic structures, Fe 3+ –O 2 - and Fe + –O 2 + , are also taken into account. The results are compared with those for the singlet and septet states calculated previously. If the value of an unknown splitting energy between u and υ orbitals is estimated to be 4,000 cm -1 or more, the singlet state composed mainly of 3 E and 3 Σ g - terms (triplet-triplet binding) becomes the lowest in energy at short distances between the heme and the oxygen molecule. Thus, the diamagnetism of oxyhemoglobin can be explained theoretically on the basis of the electronic structures of the hemes determined by the ligand field theory.

Accumulation pattern of amino acid substitutions in protein evolution

SummaryA simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This “two clock” interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.

Theoretical evaluation of transcriptional pausing effect on the attenuation in trp leader sequence

The effect of transcriptional pausing on attenuation is investigated theoretically on the basis of the attenuation control mechanism presented by Oxender et al. (Oxender, D. L., G. Zurawski, and C. Yanofsky, 1979, Proc. Natl. Acad. Sci. USA. 76:5524-5528). An extended stochastic model including the RNA polymerase pausing in the leader region is developed to calculate the probability of relative position between the RNA polymerase transcribing the trp leader sequence and the ribosome translating the transcript. The present study results in a new rationale that the transcriptional pausing site in the leader sequence makes the attenuation control both more sensitive as an on/off switch and less sensitive to variations in the concentration of cellular metabolites not connected with the need for expressing, or not expressing, the particular operon. It is also proposed that the transcriptional pausing diminishes the dependence of attenuation control characteristics on the number of nucleotides in the leader sequence. This result may be useful for understanding the attenuation control efficiencies of other amino acid leader sequences with different lengths of nucleotides.

Evolution of metabolic pathways by chance assembly of enzyme proteins generated from sense and antisense strands of pre-existing genes

In order to get an insight into the evolutionary aspect of metabolic pathways, especially of the ubiquitous glycolytic pathway, we have carried out an extensive search of sense-sense and sense-antisense similarities for enzyme proteins in the glycolytic pathway, the pentose phosphate cycle, alcohol and lactate fermentation pathways and the TCA cycle. This investigation of amino acid sequences reveals a curious pattern of similarity relations; no similarity can be found between the enzyme proteins in a section of the glycolytic pathway where the glyceraldehyde-3-phosphate or even glycerol-3-phosphate is converted into the pyruvate while many examples of sense-sense and sense-antisense similarities are found even between enzyme proteins in distant blocks, e.g. between the proteins in the TCA cycle and those in the pentose phosphate cycle, as well as between the functionally associated proteins in each of these blocks. Complementary to this characteristic pattern of amino acid sequence similarity, the search for similarities of nucleotide sequences also finds that the similarities of glycolytic enzyme genes, some sense-sense and others sense-antisense similarities, are concentrated on the nucleotide sequences of prokaryotic 16S or eukaryotic 18S ribosomal RNA gene with its flanks, although some of the copy sequences are also found in transfer RNA genes as well as in 23S or 26S ribosomal RNA gene. These results strongly suggest that the metabolic pathways have been developed by the chance assembly of enzyme proteins generated from the sense and antisense strands of pre-existing genes, e.g. the fermentation pathways and pentose phosphate cycle by the proteins from the genes of enzymes in the glycolytic pathway and the TCA cycle from all these successively increased genes, ascribing the origin of metabolic enzyme genes to the close relation between the glycolytic enzyme protein genes and the RNA gene cluster.