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T Ooi

Publications and source records attributed to T Ooi.

At least 91 records · Page 5Linked to original sources
PubMed1982

Correlation of the amino acid composition of a protein to its structural and biological characters.

Amino acid compositions of 356 proteins are expressed as points in an 18 dimensional space of 18 axes representing the contents of amino acids. The proteins are classified into four groups of intra- and extracellular enzymes and nonenzymes according to analysis of the distribution of the points. The groups have a significant correlation to four folding types of secondary structures, and extra- and intracellular proteins to those with and without disulfide bond(s), respectively. The location and function of a protein seem to determine its amino acid composition and folding type.

Amino Acids↗
PubMed1982

Cross-linking study on skeletal muscle actin: properties of suberimidate-treated actin.

Cross-linking experiments were performed on muscle skeletal actin, using imidoesters of various chain lengths. Chemical analyses on all products except one (derived from succinimidate) show evidence of the presence of intramolecular cross-links in the molecule. The detailed properties of suberimidate-treated actin (SA) are as follows: SA contains nearly 1 mol of intramolecular cross-link per mol of actin and less than 15% of intermolecularly cross-linked products. Even at a low salt concentration, SA is polymeric, exchanges slowly its bound nucleotide with free nucleotides in solution, and shows an F-actin-type CD spectrum. Electron micrographs of SA reveal that SA exists actually as fibrous polymers in solutions of low ionic strength, although the fibers seem to be less rigid than those at high salt concentration. The F-form of SA at a high salt concentration is indistinguishable from intact F-actin. SA can bind heavy meromyosin and activate the ATPase of heavy meromyosin as observed for intact F-actin. Tropomyosin binds SA only at a high salt concentration. These results show that SA possesses the properties of F-actin even in media of low salt concentration, which are favorable for depolymerization of F-actin. Thus, we may infer that the conformation of SA is frozen in the F-state of actin by the introduction of intramolecular cross-links in the protein.

Actins↗
PubMed1981

Monte Carlo simulation study of thermal fluctuations and conformational energy surface of a small protein, basic pancreatic trypsin inhibitor.

The conformation energy surface of a small protein, basic pancreatic trypsin inhibitor, is studied to characterize small-amplitude thermal fluctuations in the protein molecule. In order to see the shape of the conformational energy surface near the energy minimum point, the thermal equilibrium of the molecule is stimulated by the Monte Carlo method of Metropolis et al. From the sample of the equilibrium population, which reflects the shape of the energy surface, orthogonal directions are generated in the conformational space, and the conformational energy is actually calculated along these directions. All energy profiles along these directions are found to be approximately a parabola within the range of thermal fluctuations, which suggests the possibility of harmonic approximation to the conformational energy surface of the globular protein.

Aprotinin↗
PubMed1981

A computer method for construction of secondary structure from polynucleotide sequence. Possible structure of the bacterial replication origin.

A computer method to search the possible secondary structure of a long polynucleotide was developed. As a criterion for the stabilization of a secondary structure, free energy originating from base-pairing was employed, since the structure in solution would be at the free energy minimum. The method is summarized as follows: all possible helices are collected from a given nucleotide sequence under restrictions that the length of a helix is greater than N0 bases (e.g., four bases) and the free energy of the helix calculated according to free energies of two successive sequence-dependent basepairs is lower than E0 (e.g., -5 kcal/mol). The search of secondary structures of low free energy is performed by connecting one helix to another without allowing any base-pairing between loops. For connecting single-stranded regions, destabilizing free energy of 2--3 kcal/mol is added. The method was first applied to several tRNAs and the clover-leaf structure of tRNA was obtained as a free energy minimum. Then, possible secondary structures of the replication origin regions of the Escherichia coli and Salmonella typhimurium chromosomes were examined by the method, assuming that one of the strands in the origin region takes a specific secondary structure. The lowest-energy structure for the E. coli origin was found to be approximately identical to that for the S. typhimurium origin region.

Base Sequence↗
PubMed1981

Regeneration of ribonuclease A from the reduced protein. Isolation and identification of intermediates, and equilibrium treatment.

Reduced RNase A was reoxidized, and the incorrectly formed disulfide bonds were reshuffled to the native ones by oxidized and reduced glutathiones, as described in the first paper of this series. The intermediates in the regeneration of the disulfide bonds were trapped without any chemical modification and were fractionated on a carboxymethylcellulose column at pH 3.5 with a salt gradient. The elution curves of the partially regenerated RNase A from the carboxymethylcellulose column were obtained by measurement of the absorption at 275 nm and by determination of the SH content (of cysteine residues) and consisted of 11 fractions, G8, G7, G6, G5, G4, G3, G2, G1, G0, N, and F. Some of the fractions were isolated, and their measured molecular weights were consistent with those of monomeric RNase A. Fraction F had a molecular weight between that of the monomer and dimer, so that this fraction could not be identified. The regeneration pathway could be represented in terms of two simple reactions, RNase A(-SH) + GSSG in equilibrium or formed from RNase A(-SSG) + GSH and RNase A(-SH-SSG) in equilibrium RNase A(greater than S2) + GSH, which produced 24 monomeric intermediates (not counting the fully reduced and the native species), which differed from each other in their amino acid composition. These 24 intermediates, plus the fully reduced protein, were assigned to fractions G8--G0 (as indicated in the last column of Table I), with the aid of data from amino acid analysis, SH content, and the elution position on the carboxymethylcellulose column chromatogram. Since the regeneration reaction rapidly reached a preequilibrium among the intermediates and the fully reduced RNase A prior to the rate-limiting steps, i.e., the relative concentrations of the intermediates and fully reduced RNase A became constant with reaction time, the populations of some of the intermediates in preequilibrium were estimated by curve fitting of the elution pattern from the carboxymethylcellulose column chromatogram. The equilibrium constants among the intermediates were calculated from their populations at preequilibrium. These equilibrium constants were "extrapolated" to other intermediates whose populations could not be estimated by curve fitting, and the relative populations of all of the possible intermediates at preequilibrium were thereby represented as a function of the concentrations of reduced and oxidized glutathiones. The regeneration process was also restarted from several of the isolated intermediates, and the resulting distribution of intermediates was consistent with that from which the equilibrium constants were determined, supporting the representation of the regeneration pathways in terms of two simple reactions. Thus, the equilibrium treatment of the regeneration pathways was useful to characterize the preequilibrium state, i.e., to identify the intermediates prior to the rate-limiting steps in the pathways and to estimate their stabilities at preequilibrium at various concentrations of reduced and oxidized glutathiones.

Amino Acids↗
PubMed1980

Cross-linking study on tropomyosin.

The cross-linking reaction of alpha-tropomyosin with dimethyl adipimidate yielded a dimer of the alpha-subunit of tropomyosin as a major product, which was isolated by gel filtration on Sephadex G-150 in the presence of urea. Amino acid analyses revealed that the cross-linked alpha-tropomyosin contained about two adipimidate cross-links per molecule. Selective cleavage of the cross-linked molecule at the cysteinyl residue, Cys 190 (a single cysteinyl residue in the alpha-subunmit), gave a new band at a position corresponding to a molecular weight of 48,000 on SDS-gel electrophoresis, suggesting that the cross-links were incorporated in the N-terminal fragment. When the cross-linked molecule was cleaved with CNBr, two large fragments from residue 11 to 127, and from 142 to 281, were obtained, as in the case of the intact molecule. Therefore, it is inferred that the location of the intersubunit cross-links is in the region from residue 2 to 8 and/or from 128 to 141. These results indicate that the arrangement of alpha-subunits of tropomyosin in solution must be in parallel and in register. Although the exact positions of the reactive sites could not be determined in the present study, stereochemical examination of the coiled-coil model suggests that the most probable sites of cross-linking are Lys 5 of one subunit and Lys 7 of the other.

Amino Acid Sequence↗
PubMed1980

Prediction of the surface-interior diagram of globular proteins by an empirical method.

The number of amino acid residues in contact with a residue in a globular protein is a simple and good measure to show the relative location of the residue on the surface or in the interior of the protein. The contact number is estimated as the number of C alpha atoms within a sphere of radius r (8 A) centered at the C alpha atom of a given residue. The prediction of a diagram (the plot of the contact number against the residue number) from a given amino acid sequence may be meaningful as an alternative to the secondary-structure prediction currently performed. Parameter values are determined empirically using the observed contact numbers calculated from known structures of 39 proteins. In order to assess the real efficiency of the method, the prediction has been performed in the following way; all the proteins are divided into two groups; one group is used to derive parameter sets and the other serves to test the prediction accuracy. The test reveals that the parameter sets empirically determined are biased significantly towards the data base, the extent of which is roughly proportional to the number of parameter terms included. The results show that an adequate smoothing of a parameter set is the best way to reduce the extent of biasing towards the data base and to give the best prediction for 'unknown' proteins. The prediction accuracy finally obtained is about 0.4 (or roughly 70%), on the average, measured by the correlation coefficient between the predicted and observed diagrams. This value is of the same order as the accuracy in the current predictions of secondary structures.

Amino Acid Sequence↗
PubMed1979

Conformational stability of ribonuclease T1. II. Salt-induced renaturation.

In the presence of high concentrations of the monovalent salts, sodium chloride and potassium fluoride, disulfide-reduced RNase T1 having four cysteinyl residues intact regenerates the spectral properties characteristic of native RNase T1, e.e., the fluorescence spectrum of the aromatic side chains and the ultraviolet circular dichroism spectrum. The folding of the polypeptide chain proceeded without formation of disulfide bonds to yield an enzymatically active species having an activity toward RNA equivalent to 25% of that of the native enzyme at the same salt concentration of 2 m. Unfolding of RNase T1 by a denaturant, urea, was suppressed in the presence of salts, and the salt-induced chain folding was observed spectroscopically even in 6.9 m urea solution. The salts also induced the chain folding of disulfide reduced and modified (carboxymethylated or carboxamidomethylated) RNase T1 into the native conformation, as indicated by its spectroscopic properties, but did not restore the enzymatic activity.

Circular Dichroism↗
PubMed1979

Conformational stability of ribonuclease T1. I. Thermal denaturation and effects of salts.

The thermal transition of RNase T1 was studied by two different methods; tryptophan residue fluorescence and circular dichroism. The fluorescence measurements provide information about the environment of the indole group and CD measurements on the gross conformation of the polypeptide chain. Both measurements at pH 5 gave the same transition temperature of 56 degrees C and the same thermodynamic quantities, delta Htr (= 120 kcal/mol) and delta Str (= 360 eu/mol), for the transition from the native state to the thermally denatured state, indicating simultaneous melting of the whole molecule including the hydrophobic region where the tryptophan residue is buried. Stabilization by salts was observed in the pH range from 2 to 10, since the presence of 0.5 m NaCL caused an increase of about 5 degrees C to 10 degrees C in the transition temperature, depending on the pH. The fluorescence measurements on the RNase T1 complexed with 2'-GMP showed a transition with delta Htr =167 kcal/mol and delta Str =497 eu/mol at a transition temperature about 6 degrees C higher than that for the free enzyme. The large value of delta Htr for RNase T1 indicates the highly cooperative nature of the thermal transition; this value is much higher than those of other globular proteins. Analysis of the CD spectrum of thermally denatured RNase T1 suggests that the denatured state is not completely random but retains some ordered structures.

Circular Dichroism↗