TY - JOUR
T1 - Structural studies of kinesin-nucleotide intermediates
AU - Rosenfeld, Steven S.
AU - Correia, John J.
AU - Xing, Jun
AU - Rener, Brenda
AU - Cheung, Herbert C.
PY - 1996
Y1 - 1996
N2 - We have investigated the structural changes that occur in the molecular motor kinesin during its ATPase cycle, utilizing two bacterially expressed constructs. The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying the active site by means of sedimentation velocity, sedimentation equilibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis. While the molecular weight of monomeric and dimeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are unaffected by the nucleotide intermediate occupying the active site, significant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected. These results suggest that kinesin contains an internal 'hinge' whose flexibility varies through the course of the ATPase cycle. In prehydrolytic, 'strong' binding states, this hinge is relatively rigid, while in posthydrolytic, 'weak' binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that these two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.
AB - We have investigated the structural changes that occur in the molecular motor kinesin during its ATPase cycle, utilizing two bacterially expressed constructs. The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying the active site by means of sedimentation velocity, sedimentation equilibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis. While the molecular weight of monomeric and dimeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are unaffected by the nucleotide intermediate occupying the active site, significant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected. These results suggest that kinesin contains an internal 'hinge' whose flexibility varies through the course of the ATPase cycle. In prehydrolytic, 'strong' binding states, this hinge is relatively rigid, while in posthydrolytic, 'weak' binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that these two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.
UR - http://www.scopus.com/inward/record.url?scp=0029910778&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029910778&partnerID=8YFLogxK
U2 - 10.1074/jbc.271.47.30212
DO - 10.1074/jbc.271.47.30212
M3 - Article
C2 - 8939973
AN - SCOPUS:0029910778
SN - 0021-9258
VL - 271
SP - 30212
EP - 30221
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 47
ER -