Kyoko Ogasahara

Absence of the thermal transition in apo-α-lactalbumin in the molten globule state : A study by differential scanning microcalorimetry

To estimate the energy level of the molten globule state, the heat capacity function of apo-alpha-lactalbumin in the molten globule state has been examined using a scanning microcalorimeter at neutral pH. The results showed that the enthalpy difference between the molten globule state and presumed unfolded state by heating was almost zero at neutral pH, demonstrating that the molten globule state does not exhibit any co-operative transition upon heating. This is in agreement with the results already reported at acid pH, but is apparently in conflict with that recently reported with some assumptions at neutral pH.

Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 °C

We found that the CutA1 protein, from Pyrococcus horikoshii (PhCutA1), has an extremely high denaturation temperature (T d) of nearly 150 °C, which exceeds the highest record determined by DSC by about 30 °C. To elucidate the mechanism of the ultra‐high stability of PhCutA1, we analyzed the crystal structures of CutA1 proteins from three different sources, P. horikoshii, Thermus thermophilus, and Escherichia coli, with different growth temperatures (98, 75, and 37 °C). This analysis revealed that the remarkably increased number of ion pairs in the monomeric structure contributes to the stabilization of the trimeric structure and plays an important role in enhancing the T d, up to 150 °C, for PhCutA1.

Role of Salt Bridge Formation in Antigen-Antibody Interaction ENTROPIC CONTRIBUTION TO THE COMPLEX BETWEEN HEN EGG WHITE LYSOZYME AND ITS MONOCLONAL ANTIBODY HyHEL10

For elucidation of the role of salt bridge formation in the antigen-antibody complex, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, the structure of which has been well characterized and forms one salt bridge (Lys97 of HEL and Asp32 of HyHEL10 heavy chain variable region (VH)), was investigated. Asp32 of VH was substituted with Ala, Asn, or Glu by site-directed mutagenesis, and the interaction between HEL and the mutant fragments of the variable region of light chain was investigated by inhibition of the enzymatic activity of HEL and isothermal titration calorimetry. Inhibition assay indicated that these mutations lowered the inhibition only slightly. Thermodynamic study indicated that the negative enthalpic change in the interaction between each of the mutant variable regions of light chain and HEL was significantly increased, although the association constant was slightly decreased, suggesting that these mutations increased the entropy change upon antigen-antibody binding. These results indicate that the role of salt bridge formation in the HyHEL10-HEL interaction is to lower the entropic loss due to binding. In the mutant proteins, the numbers of residues that were perturbed structurally on binding increased, suggesting that the salt bridge suppresses excess structural movement of the antibody upon binding.

pH dependence of stability of the wild-type tryptophan synthase α-subunit and two mutant proteins (Glu49 → Met or Gln)

Abstract In order to elucidate the role of individual amino acid residues in the electrostatic interaction affecting the conformational stability of proteins, the pH dependence of stability of tryptophan synthase α-subunit of the wild-type and two mutant proteins, trp A33 (Glu49 → Met) and trp A11 (Glu49 → Gln), has been compared by means of circular dichroism measurements in the absence and presence of guanidine hydrochloride. The denaturation of the three proteins is discussed, assuming the existence of one stable intermediate. In the first denaturation step, i.e. the transition from the native to the intermediate state, the midpoint of the conformational transition in the acid region was pH 3.9 and pH 5.1 for the wild type and the trp A11 proteins, respectively; whereas, in the alkaline region the midpoint was pH 10.9 and pH 11.6 for the wild-type and the trp A11 proteins, respectively. The trp A33 protein belonged to the stronger group in stability in both the acid and alkaline regions. In the acid region in the presence of 0.4 m -guanidine hydrochloride, the trp A11 protein was more labile than the wild-type and trp A33 proteins; whereas, in the alkaline region in 0.8 m -guanidine hydrochloride, the order of stability among the three proteins was the trp A33, trp A11 and wild-type proteins at pH values between 7.5 and 9.8. In the second denaturation step, i.e. the transition from the intermediate to the completely denatured state, the order of stability among the three proteins was the trp A33, wild-type and trp A11 proteins. These results showed that a negatively charged group at position 49 served as a destabilizing factor in the alkaline region, and that the hydrophobicity of the residue at position 49 contributed to the stabilization of the conformation of the protein.

Characterization of soluble artificial proteins with random sequences

The structural and catalytic properties of two soluble random proteins, RP3‐42 and RP3‐45, of 141 amino acid residues were investigated. Although no marked secondary structure was detected by CD spectrum, sedimentation equilibrium and small‐angle X‐ray scattering studies showed that they form an oligomeric structure and are as compact as the molten globule. The random proteins have low but distinct esterase activity; the values of the second‐order rate constant for the hydrolysis of p‐nitrophenol were 0.78 and 1.39 M−1 s−1 for RP3‐42 and RP3‐45, respectively. The differences in the properties of the random and the native proteins are discussed from the evolutionary point of view.

Effect of single amino acid substitutions at the same position on stability of a two-domain protein☆

Abstract In order to elucidate the role of individual amino acid residues on the conformational stability of a protein, the stabilities of the wild-type tryptophan synthase α-subunit from Escherichia coil and its five mutant proteins substituted by single amino acid residues at the same position 49 were compared. The five mutant proteins have glutamine, methionine, valine, serine, or tyrosine in place of glutamic acid of the wild-type protein at position 49. Denaturation of these proteins, which consist of two domains, by guanidine hydrochloride can be analyzed as a two-step process. We obtained the equilibrium constants between the native and the denatured forms and between the native and the stable intermediate forms for the above six proteins in the absence of denaturant at three pH values.

Unfolding-refolding kinetics of the tryptophan synthase α subunit by CD and fluorescence measurements

To elucidate the folding mechanism of the tryptophan synthase alpha subunit from Escherichia coli, the kinetics of the unfolding-refolding were studied by peptidyl circular dichroism (CD) and aromatic fluorescence measurement at pH 7 and 25 degrees C. The reactions were induced by concentration jumps of guanidine hydrochloride (GuHCl). The results can be summarized as follows. (1) The kinetic properties of the unfolding-refolding monitored by CD at 222 nm and aromatic fluorescence coincided with each other, indicating that the changes in the secondary and tertiary structures proceed simultaneously. (2) The unfolding kinetics showed two phases in the range of final GuHCl concentration above 1.8 M. The total amplitudes in the unfolding kinetics accounted for about 100% of the total change. (3) The refolding kinetics also showed two phases in the native condition. The total amplitudes observed in the two phases accounted for only 41% of the total change in maximum, indicating the presence of an undetectable early folding intermediate in the folding process. (4) The fast phases in both the unfolding and refolding were major phases as judged by the magnitudes of the amplitudes. (5) The amplitudes in terms of the CD values at 222 nm for the undetectable early folding intermediate in the refolding kinetics showed little dependence on final GuHCl concentration in the native condition, but depended on final GuHCl concentration in the transition zone, resulting in a similar equilibrium GuHCl unfolding curve. (6) The CD spectrum in the far-UV region for the early folding intermediate was similar to that for the equilibrium unfolding intermediate. (7) It is concluded that the early folding intermediate of the alpha subunit is equivalent to the equilibrium unfolding intermediate, which is assumed to be a molten globule.

Thermal Conversion from Low- to High-activity Forms of Catalase I from Bacillus stearothermophilus

Catalase I from Bacillus stearothermophilus has the interesting property of increasing its enzyme activity on heating. It was confirmed that after heating at 70 °C for 10 min or 65 °C for 20 min, almost all the enzyme molecules were converted irreversibly to the activated form. The increase in k cat from 1400 to 3930 s−1 and the decrease in K m for H2O2 from 4.4 to 2.7 mm by heat activation indicate changes in the kinetic property of the enzyme molecule. Therefore, it follows that catalase I has two active forms, a high-activity form and a low-activity form. The heat activation process followed the first-order kinetics with an activation enthalpy (ΔH*) of 191 kJ/mol while the heat denaturation process had a ΔH* of 545 kJ/mol. The CD spectra of the two enzyme forms had small but marked differences. The conversion of the low-activity form to the high-activity form was an endothermic process with aT m of 56 °C, which is much lower than that of the heat denaturation (T m = 76 °C), and the enthalpy change for the transition was only 5% of that for the denaturation. It has to be noted that the high-activity form of the enzyme was converted back to a low-activity form through the process of denaturation, refolding, and reconstitution with heme. In addition, the newly obtained low-activity form was brought to a high-activity form by heating. These results suggest that the native state of catalase I has two active conformations that are roughly the same but not identical and are separated by a high energy barrier.

Effect of amino acid substitutions on conformational stability of a protein.

This paper reviews studies on thermostable proteins from thermophilic bacteria and on mutant proteins of human hemoglobin, tryptophan synthase alpha-subunit of E. coli, T4 phage lysozyme, and phage lambda repressor with respect to the role of the constituting amino acid residues in stabilization of conformation. The stability of a protein is easily affected by single amino acid substitutions, by which the protein undergoes change(s) of one or more of the following: a hydrogen bond, a salt bridge, a hydrophobic interaction, the volume of the residue, a disulfide bond, or the relative position of two aromatic rings.

Comparison of denaturation of tryptophan synthase α-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase alpha subunit have been compared by circular dichroism measurements. The three alpha subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (alpha-2 domain) and the N-terminal domain comes from S. typhimurium (alpha-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 degrees C showed that the alpha-2 domain of S. typhimurium was more stable than the alpha-2 domain of E. coli, but the alpha-1 domain of S. typhimurium was less stable than the alpha-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 degrees C and coincided with that obtained from guanidine hydrochloride-induced denaturation.