Dual roles of Gln137 of actin revealed by recombinant human cardiac muscle alpha-actin mutants.
The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln(137) to understand the relationships between two activities: the conformational changes relevant to the G- to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln(137) in these activities, we characterized Gln(137) mutants of human cardiac muscle alpha-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the gamma-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln(137) plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.
Iwasa
Mitsusada
M
ERATO Actin Filament Dynamics Project, Japan Science and Technology Agency, c/o RIKEN SPring-8 Center, Sayo, Hyogo, Japan.
Maeda
Kayo
K
Narita
Akihiro
A
Maéda
Yuichiro
Y
Oda
Toshiro
T
D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D005786:Gene Expression Regulation; D005973:Glutamine / Q000737:chemistry*; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry
D000199:Actins; D011994:Recombinant Proteins; D005973:Glutamine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate