ATP-induced transconformation of myosin revealed by determining three-dimensional positions of fluorophores from fluorescence energy transfer measurements.
The method of fluorescence resonance energy transfer (FRET) is one of the most important techniques for measuring the distance between two fluorophores and for detecting the changes in protein structure under physiological conditions. The use of green fluorescent protein is also a powerful technology that has been used to elucidate dynamic molecular events. From these we have developed a novel method to determine the three-dimensional positions of fluorophores by combining the FRET data and other structural information available. Using this method, we could determine the ATP-induced changes of three-dimensional structure of truncated Dictyostelium myosin in solution. The myosin structure with ADP in solution was found to be similar to that of the crystal structure of MgADPBeFx-bound truncated Dictyostelium myosin (type I structure), whereas myosin with ATP in solution was similar to the crystal structure of MgAdPVi-bound one (type II structure). However, the crystal structure of MgADP-bound scallop myosin (type III structure) could not be explained by any of our FRET data under various conditions. This indicates that the type III crystal structure might represent a transient intermediate conformation that could not be detected using fluorescence energy transfer.
Yasunaga
T
T
Department of Physics, School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo, Tokyo 113-0033, Japan.
Suzuki
Y
Y
Ohkura
R
R
Sutoh
K
K
Wakabayashi
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000465:Algorithms; D000818:Animals; D003460:Crystallization; D004023:Dictyostelium / Q000737:chemistry; D004735:Energy Transfer; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D021621:Imaging, Three-Dimensional; D008164:Luminescent Proteins; D008274:Magnesium / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008958:Models, Molecular; D008974:Mollusca / Q000737:chemistry; D009218:Myosins / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D011487:Protein Conformation
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D009218:Myosins; D008274:Magnesium