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M Oobatake

Publications and source records attributed to M Oobatake.

34 records · Page 2Linked to original sources
PubMed1994

De novo design and creation of a stable artificial protein.

Protein de novo design has been performed, as an exercise of the inverse folding problem. A beta/alpha-barrel protein was designed and synthesized using the Escherichia coli expression system for the structural characterization. A tertiary model with a two-fold symmetry was built, based upon the geometrical parameters extracted from X-ray crystal structures of several beta/alpha-barrel proteins. Amino acid frequencies at each position on the alpha- and beta-structures were investigated, and an amino acid sequence with 201 residues was designed. The associated gene was chemically synthesized and the fusion protein with human growth hormone was expressed in Escherichia coli. The purified protein after being cleaved and refolded was found to be stable and globular with the large amount of secondary structures. However, it has similar characteristics to the molten globules of natural proteins, with loose packing of side-chains. The approach for the tight packing is discussed.

Amino Acid Sequence↗
PubMed1993

Hydration and heat stability effects on protein unfolding.

In summary, the thermal denaturation of proteins has been elucidated in terms of the chain free energy and the hydration free energy as follows. (1) Method to calculate the unfolding free energy. The free energy of unfolding consists of two contributions: the hydration around the molecule, and the intramolecular interactions. A method to calculate the free energy of hydration from the accessible surface area (ASA) of the constituent atomic groups in a protein has been developed. This assumes a proportionality between the free energy and the ASA, where the proportional constants were determined by least-squares fitting to the experimentally derived thermodynamic data on small molecules. Similarly, the free energy of unfolding for the chain in vacuo can be also calculated from the ASA, using the unfolding thermodynamics derived from the experimental data of the ten proteins. (2) Thermodynamics of protein unfolding predicted from the three-dimensional structures and from the amino acid content in proteins. First, our method is applied to predict the thermodynamics of protein unfolding from the X-ray structure. The predicted values of four test proteins agree well with the experimentally derived values. It also accounts for the temperature dependence of the free energy and of the enthalpy upon unfolding for 14 proteins. Second, this method is applied to the helix-coil transition of short peptides of poly(L-Ala)20 and Ac-(AAAAK)3A-NH2. The calculated enthalpy change is close to the experimental values for poly-L-Lys and poly-L-Glu. Since delta Hcu at 25 degrees C significantly contributes to delta Gu, the helix formation is enthalpy-driven through interactions in the chain. Third, the method is applied to predict the unfolding thermodynamics of a globular protein from its amino acid content. It also accounts for the temperature dependence of the free energy of unfolding for the 14 proteins. The agreement between the experimental and the calculated values by this method for the 14 proteins is not so different from those obtained with the three-dimensional structures. Fourth, the values of delta Cpu for 14 proteins may be closely approximated to the predicted values of delta Cp,hu. The delta Cp,hu value in a protein consists of the major contribution from the hydrophobic and the aromatic residues, and the minor one from the hydrophilic residues. (3) Dominant free energies in protein folding.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

pH-dependent thermostabilization of Escherichia coli ribonuclease HI by histidine to alanine substitutions.

Thermal stabilities of mutant ribonuclease HI proteins from Escherichia coli, in which each of five histidine residues was replaced with alanine, were examined at various pHs. Increases in the Tm values were observed at pH 3.0 for four of the mutant proteins, in which each of the four histidine residues exposed to the solvent was mutated, as compared to the Tm of the wild-type protein. The thermostabilization of three of the mutant proteins was dependent on pH, and only observed at low pH. The thermostabilizing effects of the His-->Ala substitutions were cumulative. The temperature of the midpoint of the transition in the thermal unfolding curves, Tm, of the most stable mutant enzyme, in which His 62, His 83, His 124, and His 127 were replaced by Ala, was 5.5 degrees C higher than that of the wild-type enzyme at pH 3.0. The stability of the wild-type protein decreased as the pH was lowered below pH 4, a condition favoring the protonation of carboxyl groups, probably due to unfavorable electrostatic interactions introduced by the increase in positive charges on the protein. Since imidazole groups are positively charged at pH 3.0, it seems likely that thermal stabilization at pH 3.0 by a His-->Ala substitution would be the result of a reduction in such unfavorable electrostatic interactions. These results suggest that amino acid substitutions that cause a decrease in the number of positive charges on the surface of a protein can be used as a general strategy to enhance protein stability at pH values below pH 4.

Alanine↗
PubMed1992

Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart.

Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M. (1992) J. Biol. Chem. 267, 10184-10192). The identity in the amino acid sequences of these enzymes is 52%. As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability. A variety of mutant proteins of E. coli RNase HI were constructed and analyzed for protein stability. In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T. thermophilus RNase H. Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability. Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative. Replacement of all four regions (R4-R7) gave the most stable mutant protein. The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E. coli RNase HI. These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner.

Amino Acid Sequence↗
PubMed1992

Design and synthesis of an alpha-helical peptide containing periodic proline residues.

A thirty-residue peptide (PERI COIL-1) has been designed with a new type of alpha-helical structure, which is capable of folding into an amphiphilic helix bending at 4 periodic prolines in the sequence. Two such helices should form a dimer by supercoiling about one another in an antiparallel direction in the design. With this arrangement, close packing between them is maintained through the hydrophobic interaction pattern called 'leucine zipper'. PERI COIL-1 has been obtained by solid-phase peptide synthesis, and characterized by circular dichroic spectroscopy, sedimentation equilibrium experiments and NMR. The result of the analyses shows that it preferentially forms a helical tetramer in aqueous solution.

Amino Acid Sequence↗
PubMed1991

Prediction of the thermodynamics of protein unfolding: the helix-coil transition of poly(L-alanine).

The method given earlier for predicting the thermodynamics of protein unfolding from the x-ray structure of a protein is applied here to the poly(L-alanine) helix. First, the fitting parameters derived earlier from a data base of 10 proteins were used to predict the unfolding thermodynamics of 4 other proteins. The agreement between the observed and predicted values is comparable to that found for the 10 proteins studied initially. Next, the temperature dependences of the Gibbs energy and enthalpy changes for unfolding of bacteriophage T4 lysozyme were predicted and compared with data in the literature. The predicted and observed temperature dependences are similar and the predicted results indicate that cold denaturation should be observed at low temperatures, as observed recently for a T4 lysozyme mutant. The fitting parameters derived from thermodynamic data for protein unfolding and for hydration of model compounds were used to predict the unfolding thermodynamics of the poly(L-alanine) helix. The results predict that helix formation is enthalpy-driven, and the predicted enthalpy change for unfolding (0.86 kcal per mol per residue) is close to the value found in a recent calorimetric study of a 50-residue alanine-rich helix.

Kinetics↗
PubMed1988

Effects of hydrated water on protein unfolding.

The conformational stability of a protein in aqueous solution is described in terms of the thermodynamic properties such as unfolding Gibbs free energy, which is the difference in the free energy (Gibbs function) between the native and random conformations in solution. The properties are composed of two contributions, one from enthalpy due to intramolecular interactions among constituent atoms and chain entropy of the backbone and side chains, and the other from the hydrated water around a protein molecule. The hydration free energy and enthalpy at a given temperature for a protein of known three-dimensional structure can be calculated from the accessible surface areas of constituent atoms according to a method developed recently. Since the hydration free energy and enthalpy for random conformations are computed from those for an extended conformation, the thermodynamic properties of unfolding are evaluated quantitatively. The evaluated hydration properties for proteins of known transition temperature (Tm) and unfolding enthalpy (delta Hm) show an approximately linear dependence on the number of constituent heavy atoms. Since the unfolding free energy is zero at Tm, the enthalpy originating from interatomic interactions of a polypeptide chain and the chain entropy are evaluated from an experimental value of delta Hm and computed properties due to the hydrated water around the molecule at Tm. The chain enthalpy and entropy thus estimated are largely compensated by the hydration enthalpy and entropy, respectively, making the unfolding free energy and enthalpy relatively small. The computed temperature dependences of the unfolding free energy and enthalpy for RNase A, T4 lysozyme, and myoglobin showed a good agreement with the experimental ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics↗
PubMed1988

Characteristic thermodynamic properties of hydrated water for 20 amino acid residues in globular proteins.

Thermodynamic properties associated with hydrated water of proteins of known three-dimensional structure were computed and average values of hydration free energy, enthalpy, and heat capacity of unfolding for every amino acid residue were obtained. Each amino acid residue had characteristic values; in particular, the quantities for a side chain reflected the character of the amino acid, while those for the main chain were more or less the same except for glycine, alanine, and proline. The major contribution to the quantities was from the end group(s) of a side chain. The following interesting features were found. 1) The hydration quantity of unfolding derived from the native and extended conformations for a protein was approximately equal to the sum of the corresponding average quantities of component amino acid residues in the protein. 2) The profile of a quantity such as hydration free energy of unfolding along the sequence computed from the accessible surface areas of the native and extended conformations showed a strong correlation with the profile obtained by allocating the average value for the amino acid residue at every position on the sequence. The correlation coefficients between two profiles for unfolding quantities of hydration, i.e., free energy, enthalpy, heat capacity, and free energy of side chain are 0.72, 0.62, 0.80, and 0.75, respectively. Thus, every amino acid residue in the native conformation of a globular protein seems to be located in such a position that a thermodynamic quantity for each residue is approximately equal to its average value.

Amino Acids↗
PubMed1988

Intermolecular interactions between protein and other molecules including hydration effects.

The structural aspects of protein functions, e.g., molecular recognition such as enzyme-substrate and antibody-antigen interactions, are elucidated in terms of dehydration and atomic interactions. When a protein interacts with some target molecule, water molecules at the interacting regions of both molecules are removed, with loss of the hydration free energy, but gaining atomic interactions between atoms of the contact sites in both molecules. The free energies of association originating from the dehydration and interactions between the atoms can be computed from changes in the accessible surface areas of the atoms involved. The free energy due to interactions between atomic groups at the contact sites is estimated as the sum of those estimated from the changes in the accessible surface area of 7 atomic groups, assuming that the interactions are proportional to the change of the area. The chain enthalpies and entropies evaluated from experimental thermodynamic properties and hydration quantities at the standard temperature for 10 proteins were available to determine the proportional constants for the atomic groups. This method was applied to the evaluation of association constants for the dimerization of proteins and the formation of proteolytic enzyme-inhibitor complexes, and the computed constants were in agreement with the experimental ones. However, the method is not accurate enough to account quantitatively for the change in the thermal stability of mutants of T4 lysozyme. Nevertheless, this method provides a way to elucidate the interactions between molecules in solution.

Proteins↗
PubMed1987

Accessible surface areas as a measure of the thermodynamic parameters of hydration of peptides.

A method is described for the inclusion of the effects of hydration in empirical conformational energy computations on polypeptides. The free energy of hydration is composed of additive contributions of various functional groups. The hydration of each group is assumed to be proportional to the accessible surface area of the group. The constants of proportionality, representing the free energy of hydration per unit area of accessible surface, have been evaluated for seven classes of groups (occurring in peptides) by least-squares fitting to experimental free energies of solution of small monofunctional aliphatic and aromatic molecules. The same method has also been applied to the modeling of the enthalpy and heat capacity of hydration, each of which is computed from the accessible surface area.

Amino Acids↗
PubMed1985

Molecular orientation of plastocyanin on spinach thylakoid membranes as determined by acetylation of lysine residues.

To reveal the molecular orientation of plastocyanin (PC) on spinach thylakoid membranes, the position of Lys residues modified by acetic anhydride was compared between thylakoid-bound PC and the isolated one. Digestion of the isolated PC by a trypsin yielded a peptide map with seven spots prior to the acetylation of the protein; none of the spots appeared after the isolated PC was acetylated. On the other hand, there were two spots on the peptide map of the PC acetylated when it was bound to the thylakoids. Those spots were revealed by their amino acid compositions to correspond to the peptide fragments Phe 82-Lys 95 and Val 96-Asn 99. Thus, the Lys residues 81 and 95 of the thylakoid-bound PC were not acetylated. These results suggest that the PC molecule binds to the thylakoids with a specific region including the Lys's 81 and 95 in contact with the membranes. The Lys's 81 and 95 are located near Tyr 83, which has been thought to be the delivery site of electrons from the Cu2+ center.

Acetylation↗
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↗
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↗
PubMedDate not supplied

Thermodynamic databases for proteins and protein-nucleic acid interactions.

Thermodynamic data regarding proteins and their interactions are important for understanding the mechanisms of protein folding, protein stability, and molecular recognition. Although there are several structural databases available for proteins and their complexes with other molecules, databases for experimental thermodynamic data on protein stability and interactions are rather scarce. Thus, we have developed two electronically accessible thermodynamic databases. ProTherm, Thermodynamic Database for Proteins and Mutants, contains numerical data of several thermodynamic parameters of protein stability, experimental methods and conditions, along with structural, functional, and literature information. ProNIT, Thermodynamic Database for Protein-Nucleic Acid Interactions, contains thermodynamic data for protein-nucleic acid binding, experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data have been incorporated into 3DinSight, an integrated database for structure, function, and properties of biomolecules. A WWW interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. These thermodynamic databases, together with structural databases, help researchers gain insight into the relationship among structure, function, and thermodynamics of proteins and their interactions, and will become useful resources for studying proteins in the postgenomic era.

DNA↗