Kinetic Characterization of the Function of Myosin Loop 4 in the Actin-Myosin Interaction

@article{citeulike:2440623, abstract = {Abstract: Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (AL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.}, address = {Department of Biochemistry, E\"{o}tv\"{o}s University, Institute of Biology, Budapest, H-1117, Hungary, Institute of Mechanics, Lomonosov Moscow State University, Vorobjovy Gory, Moscow 119192, Russia, and Department of Biophysics, University of P\'{e}cs, Faculty of Medicine, P\'{e}cs, H-7624 Hungary}, author = {Gyimesi, M. and Tsaturyan, A. K. and Kellermayer, M. S. Z. and Malnasi-Csizmadia, A. }, citeulike-article-id = {2440623}, doi = {10.1021/bi701554a}, journal = {Biochemistry}, keywords = {fluorescent, kinetics, motor, mutation, myosin, trp}, month = {January}, number = {1}, pages = {283--291}, posted-at = {2008-02-28 09:49:37}, priority = {2}, title = {Kinetic Characterization of the Function of Myosin Loop 4 in the Actin-Myosin Interaction}, url = {http://dx.doi.org/10.1021/bi701554a}, volume = {47}, year = {2008} }

TY - JOUR ID - citeulike:2440623 L3 - citeulike-article-id:2440623 TI - Kinetic Characterization of the Function of Myosin Loop 4 in the Actin-Myosin Interaction JF - Biochemistry VL - 47 IS - 1 SP - 283 EP - 291 AD - Department of Biochemistry, Eötvös University, Institute of Biology, Budapest, H-1117, Hungary, Institute of Mechanics, Lomonosov Moscow State University, Vorobjovy Gory, Moscow 119192, Russia, and Department of Biophysics, University of Pécs, Faculty of Medicine, Pécs, H-7624 Hungary N2 - Abstract: Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (AL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states. KW - fluorescent KW - kinetics KW - motor KW - mutation KW - myosin KW - trp AU - Gyimesi, M AU - Tsaturyan, AK AU - Kellermayer, MSZ AU - Malnasi-Csizmadia, A PY - 2008/01/08/ UR - http://dx.doi.org/10.1021/bi701554a ER -