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T Ooi

Publications and source records attributed to T Ooi.

At least 109 records · Page 6Linked to original sources
PubMed1978

Flexibility of bovine pancreatic trypsin inhibitor.

The native conformation of a protein may be expressed in terms of the dihedral angles, phi's and psi's for the backbone, and kappa's for the side chains, for a given geometry (bond lengths and bond angles). We have developed a method to obtain the dihedral angles for a low-energy structure of a protein, starting with the X-ray structure; it is applied here to examine the degree of flexibility of bovine pancreatic trypsin inhibitor. Minimization of the total energy of the inhibitor (including nonbonded, electrostatic, torsional, hydrogen bonding, and disulfide loop energies) yields a conformation having a total energy of -221 kcal/mol and a root mean square deviation between all atoms of the computed and experimental structures of 0.63 A. The optimal conformation is not unique, however, there being at least two other conformations of low-energy (-222 and -220 kcal/mol), which resemble the experimental one (root mean square deviations of 0.66 and 0.64 A, respectively). These three conformations are located in different positions in phi, psi space, i.e., with a total deviation of 81 degrees, 100 degrees and 55 degrees from each other (with a root mean square deviation of several degrees per dihedral angle from each other). The nonbonded energies of the backbones, calculated along lines in phi, psi space connecting these three conformations, are all negative, without any intervening energy barriers (on an energy contour map in the phi, psi plane). Side chains were attached at several representative positions in this plane, and the total energy was minimized by varying the kappa's. The energies were of approximately the same magnitude as the previous ones, indicating that the conformation of low energy is flexible to some extent in a restricted region of phi, psi space. Interestingly, the difference delta phi i+1 in phi i+1 for the (i + 1)th residue from one conformation to another is approximately the same as -delta psi i for the ith residue; i.e., the plane of the peptide group between the ith and (i + 1)th residues re-orient without significant changes in the positions of the other atoms. The flexibility of the orientations of the planes of the peptide groups is probably coupled in a cooperative manner to the flexibility of the positions of the backbone and side-chain atoms.

Animals↗
PubMed1978

Tropomyosin fragments obtained by tryptic digestion.

Rabbit skeletal alpha-tropomyosin, separated by hydroxyapatite chromatography, was treated with trypsin (1/100 wt/wt) at 0 degrees C for 24 h. Trypsin-resistant fragments of tropomyosin were separated into the precipitate and supernatant fractions at pH 4.3 in 1 M KCl, and these were subjected to QAE-Sephadex A50 column chromatography for further purification. SDS-gel electrophoresis showed 16,000 and 14,000 dalton bands for the supernatant (s-fragment) and an 11,500 dalton band for the precipitate (p-fragment). We obtained a 13,500 dalton chain (13,500 dalton fragment) in addition to the s- and p-fragments upon treatment with more dilute trypsin (1/500 wt/wt) for 48 h at 0 degrees C. Both the p- and 13,500 dalton fragment had the same C-terminal portion as intact alpha-tropomyosin, and could form an intra-chain disulfide bond on oxidation. Therefore, these two fragments were deduced to be polypeptides from some points on the N-terminal side of Cys 190 to the intact C-terminal. The s-fragment, on the other hand, did not contain any cysteine, Phe, or His residues according to amino acid analysis, suggesting that the fragment is derived from the N-terminal side from Cys 190. Tentative assignment of the fragments was carried out by amino acid analysis, and C- and N-terminal determination. The p-, s-, and 13,500 dalton fragments appear to be in coiled-coil form in solution, having alpha-helical contents of 77,71, and 64%, respectively, and are able to interact with intact tropomyosin to reduce the viscosity of tropomyosin solution. The s-, p-, and 13,500 dalton fragments have little binding capacity individually to troponin, but the mixture, i.e., the s- and p-fragments, the 13,500 dalton fragment and the N-chain, which was obtained by cleavage at Cys 190, showed clear binding with troponin independent of Ca2+ in solution as detected by gel electrophoresis. The p-fragment showed some binding to troponin, since cross-linkage to troponin was possible by treatment with dimethyl suberimidate. From the result, it can be inferred that the troponin binding regions in tropomyosin are located on both sides of Cys 190, where trypsin attacks more easily than at other parts of the molecule, leaving two trypsin-resistant fragments.

Amino Acid Sequence↗
PubMed1977

Troponin binding region of tropomyosin.

Four fragments of alpha-tropomyosin were prepared by specific cleavage at the Cys 190 by 2-nitro-5-thiocyanobenzoic acid and by tryptic digestion. These fragments were called the N-chain corresponding to residue 1 to 189 of the original chain, the C-chain from 190 to 284, the s-fragment from 13 to 149 and/or 128, and the p-fragment from 183 to 284, respectively. Fragments individually have little binding capacity to troponin as shown by gel electrophoresis. But a new band of the complex with troponin was detected using mixtures of the fragments, one from the N-terminal side and the other from the C-terminal side, i.e., the N- and C-chains, the s- and p-fragments, the N-chain and the p-fragment, and the s-fragment and the C-chain. Therefore, the troponin binding region of tropomyosin is thought to be located between residues 150 and 190.

Binding Sites↗
PubMed1977

A circular dichroic spectral study on disulfide-reduced pancreatic ribonuclease A and its renaturation to the active enzyme.

Disulfide-reduced RNase A, which could be reoxidized to give the native enzyme, was shown to have a CD spectrum quite different from that of the native enzyme or a random coil. Disulfide-reduced and fully cysteine-S-carboxamidomethylated RNase A because the derivative was stable and gave a spectrum identical to that of reduced RNase A. Curve-fitting analyses showed the presence of 14% alpha-helix and 25% beta-structure in this open chain derivative of RNase A. The time dependence of CD spectra during the oxidative renaturation of reduced RNase A was analyzed and changes in alpha-helical and beta-structure contents during the reaction were estimated. It was shown that the change in the content of beta-structure was slower than that of alpha-helix content and approximately paralleled the appearance of the enzymatic activity.

Amino Acids↗
PubMed1976

Properties of non-polymerizable tropomyosin obtained by carboxypeptidase A digestion.

Tropomyosin digested with carboxypeptidase A [EC 3.4.12.2] (CTM) shows a lower viscosity than the undigested protein in solution. From the relation between the viscosity decrease and the amount of amino acids liberated from the carboxyl terminus during this digestion, it is inferred that loss of the tri-peptide-Thr-Ser-Ile from the C-terminus is responsible for the decrease in viscosity. The secondary structure of -TM was not affected by the digestion according to circular dichroic measurements. The viscosity of CTM did not increase in methanol-water mixtures, whereas that of tropomyosin increased markedly. These results indicate that polymerizability was lost upon the removal of a small peptide from the C-terminus without change in the secondary structure. A decrease in the viscosity of tropomyosin solutions was observed on the addition of CTM, indicating that CTM interacts with intact tropomyosin. The dependence of the viscosity decrease on the amount of CTM showed that CTM binds tropomyosin in a one-to-one ratio as a result of end-to-end interaction. Since paracrystals having a 400 A repeated band structure could be grown in the presence of Mg ions at neutral pH, side-by-side interactions in CTM molecules remain intact, even though polymerizability is lost. The disc gel electrophoretic pattern showed that troponin could bind to CTM, but no increase in viscosity due to the complex was observed in solution. That is, the C-terminal part of tropomyosin is not required for the formation of the complex. The amount of CTM bound to F-actin was less than half of that bound to undigested tropomyosin, and could be reduced to one-tenth by a washing procedure. In the presence of troponin, however, the amount recovered to the level of tropomyosin normally bound to F-actin. Therefore, it is concluded that troponin is bound in the middle of the tropomyosin molecule and strengthens the binding of tropomyosin to F-actin.

Actins↗
PubMed1975

Similarities and differences of the alpha and beta components of tropomyosin.

Rabbit skeletal tropomyosin was separated into two components, alpha and beta, by CM cellulose column chromatography in the presence of urea. The two components are apparently different from TN-T, since, 1) upon addition of the components to F-actin solutions, they increase the degree of flow birefringence delta n, while TN-T does not, 2) the reduced mean residue elipticities [theta] at 220 nm are about 2.5-fold higher than for TN-T, and they contain no proline. These features are similar to those of intact tropomyosin, but the two components are not identical for the following reasons; 1) leucine is the C-terminus of the beta component and isoleucine is the C-terminus of the alpha component, 2) the beta component has a lower helicity and a somewhate lower capacity to increase delta n of F-actin solutions than the alpha component, and 3) the beta component has a higher content of glutamic acid and methionine than the alpha component. The two components can be crystallized into paracrystals in the presence of magnesium. Electron micrographs of the paracrystals of both components show a band pattern with 400 A periodicity. Bovine cardiac tropomyosin migrates on SDS gels as two poorly resolved bands, which could be separated by CM cellulose column chromatography. The C-terminus of the slower moving component was leucine, and that of the faster moving component was isoleucine, corresponding to the beta and alpha components of skeletal tropomyosin.

Actins↗
PubMed1975

Non-polymerizable tropomyosin and control of the superprecipitation of actomyosin.

Non-polymerizable tropomyosin was prepared by the digestion of several C-terminal residues of tropomyosin with carboxypeptidase A [EC 3.4.12.2]. The intrinsic viscosity and molecular weight of the non-polymerizable tropomyosin were almost the same as those of untreated tropomyosin. Like untreated tropomyosin, the non-polymerizable tropomyosin in combination with troponin repressed the superprecipitation of actomyosin in the absence of calcium, while this repression was released by addition of calcium. However, the curve representing the superprecipitation rate as a function of pCa was less steep than that found with actomyosin containing untreated tropomyosin: in the former case, the rate increased to a plateau over about 2 pCa units, while in the latter case, it did so over about 1 pCa unit. These experimental results provide evidence that the "co-operation" in the regulation mechanism of skeletal muscle contraction, which is indicated by the steep curve of the contraction versus pCa relation, is mediated by tropomyosin-tropomyosin interaction along the thin filament.

Actomyosin↗