Kenshu Kamiya

Interaction between the antigen and antibody is controlled by the constant domains: Normal mode dynamics of the HEL–HyHEL-10 complex

The antigen binding fragment (Fab) of a monoclonal antibody (HyHEL‐10) consists of variable domains (Fv) and constant domains (CL–CH1). Normal modes have been calculated from the three‐dimensional structures of hen egg lysozyme (HEL) with Fab, those of HEL with Fv, and so on. Only a small structural change was found between HEL–Fab and HEL–Fv complexes. However, HEL–Fv had a one order of magnitude lower dissociation constant than HEL–Fab. The Cα fluctuations of HEL–Fab differed from those of HEL–Fv with normal mode calculation, and the dynamics can be thought to be related to the protein–protein interactions. CL–CH1 may have influence not only around local interfaces between CL–CH1 and Fv, but also around the interacting regions between HEL and Fv, which are longitudinally distant. Eighteen water molecules were found in HEL–Fv around the interface between HEL and Fv compared with one water molecule in HEL–Fab. These solvent molecules may occupy the holes and channels, which may occur due to imperfect complementarity of the complex. Therefore, the suppression of atomic vibration around the interface between Fv and HEL can be thought to be related to favorable and compact interface formation by complete desolvation. It is suggested that the ability to control the antigen–antibody affinity is obtained from modifying the CL–CH1. The second upper loop in the constant domain of the light chain (UL2–CL), which is a conserved gene in several light chains, showed the most remarkable fluctuation changes. UL2–CL could play an important role and could be attractive for modification in protein engineering.

Non-Arrhenius temperature dependence of the rate constant for the H + H2S reaction

Abstract The rate constants for the H(2S) + H2S reaction were measured behind reflected shock waves (T = 1053–2237 K) and at 293 K, in which H(2S) atoms were generated by the ArF-laser photolysis of H2S. The resulting rate constant, k = (1.96 ± 0.7) × 10−17T2.1±0.3 exp[− (352 ± 84) / T] cm3 molecule−1 s−1, showed a strong non-Arrhenius dependence on the temperature. This dependence is explained by a conventional transition-state theory combined with a quantum-mechanical calculation of the transition state at the PMP4(SDTQ)6-311G**//MP2/6-31G** level.

Algorithm for normal mode analysis with general internal coordinates

A technique for performing normal vibrational analysis for biological macromolecules using general internal coordinates is proposed. The technique is based on the conventional algorithm for calculating the second derivatives of potential and kinetic energies using intramolecular dihedral angles, intermolecular translation, and rotation as variables [Braun, W. et al., J Phys Soc Jpn 1984, 53, 3269]. We extend the algorithm to include more general internal coordinates, bond stretching, angle bending, and so forth, without assuming two‐body interactions. The essential point is the separation of the variables for potential functions and vibrational analysis. With our technique, we can arbitrarily choose any combination of internal coordinates as variables, free from the functional form of potential energy. We can analyze complex systems such as a multiple molecular system including solvents or a transition state of chemical reactions. In addition, mixed use of the potentials of molecular mechanics and quantum chemistry is possible.

Dynamic character of the complex of human blood coagulation factor VIIa with the extracellular domain of human tissue factor: a normal mode analysis.

As an attempt to investigate the dynamic interactions between plasma serine protease, coagulation factor VIIa (VIIa) and its cofactor, tissue factor (TF), we performed normal mode analysis (NMA) of the complex of VIIa with soluble TF (the extracellular part of TF; sTF). We compared fluctuations of Cα atoms of VIIa or sTF derived from NMA in the VIIa‐sTF complex with those of VIIa or sTF in an uncomplexed condition. The atomic fluctuations of the Cα atoms of sTF complexed with VIIa did not significantly differ from those of sTF without VIIa. In contrast, the atomic fluctuations of VIIa complexed with sTF were much smaller than those of VIIa without sTF. These results suggest that domain motions of VIIa molecule alone are markedly dampened in the VIIa‐sTF complex and that the sTF molecule is relatively more rigid than the VIIa molecule. This may indicate functions of TF as a cofactor.

Factor IX Bm Kiryu: a Val‐313‐to‐Asp substitution in the catalytic domain results in loss of function due to a conformational change of the surface loop: evidence obtained by chimaeric modelling

Summary. Factor IX Kiryu is a naturally occurring mutant of factor IX that has 2.5% coagulant activity, even though normal plasma levels of factor IX antigen are detected. Factor IX Kiryu was purified from a patients plasma by immunoaffinity chromatography with a calcium‐dependent anti‐factor IX monoclonal antibody column. It was cleaved normally by factor XIa in the presence of Ca2+, yielding a two‐chain factor IXa. However, the resulting factor IXa showed only 1.5% of the normal factor IXa in terms of factor X activation in the presence of factor VIII, phospholipids, and Ca2+, and had 20% of the normal esterase activity for Z‐Arg‐p‐nitrobenzyl ester. Therefore factor IXa Kiryu showed the defect of the catalytic triad or primary substrate binding site as well as defective interaction with factors VIII/X. Single‐strand conformational polymorphism analysis and DNA sequencing of the amplified DNA revealed a missense point mutation, a T‐to‐A substitution at nucleotide number 31 059 of the factor IX Kiryu gene. This mutation resulted in the amino acid substitution of Val‐313 by Asp in the catalytic domain. Restriction enzyme analysis of the amplified DNA showed that the mutation was inherited from the patients mother. The chimaeric method was employed to construct a model of the serine protease domain of factor IXa, and the resultant model suggested that the Val‐313 to Asp substitution altered the conformation of the substrate‐binding site. These data combined with our previous findings on a Gly‐311‐to‐Glu mutant of factor IX suggest that the loop conformation from Gly‐311 to Arg‐318 is important for the expression of coagulant activity.