Evidence for existence of multiple conformations of kinesin and ncd motor domains in solution revealed by 31P-NMR of the tightly bound ADP.
The motor domains of kinesin and ncd contain ADP bound tightly at the enzymatic active sites even after purification. The microenvironments surrounding the phosphates of the tightly bound ADP molecules in these motor domains were studied using 31P-NMR. The alpha-phosphate and beta-phosphate of the tightly bound ADP molecules gave 31P-NMR signals at positions shifted to higher and lower field, respectively, from those of free MgADP in solution. On the other hand, the peak areas of the signals of the alpha-phosphates and beta-phosphates of bound ADP were much smaller than those of free MgADP. The temperature dependence of these signals was investigated to reveal that each signal of bound MgADP exhibited a large temperature dependence in the peak area from 0 to 20 degrees C with both proteins. This result implied that these motor domains would take multiple conformations in solution; one of these conformations gives the signals of bound MgADP observed by 31P-NMR as above, while another makes the signals of MgADP very broad and, hence, invisible in the 31P-NMR spectra. A possible reason for invisible signals would be strong chemical shift anisotropy effects of the phosphorus nuclei. In addition, we found a difference between kinesin and ncd motor domains in the chemical shift of the alpha-phosphate of bound MgADP, indicating that the electrostatic or magnetic microenvironments of this site of these motor domains differed from each other.
Suzuki
Y
Y
Biotechnology Research Center, University of Tokyo, Japan.
Tanokura
M
M
Shimizu
T
T
D000244:Adenosine Diphosphate / Q000378:metabolism*; D029721:Drosophila Proteins; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010759:Phosphorus Isotopes; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions; D013696:Temperature
D029721:Drosophila Proteins; D010759:Phosphorus Isotopes; D012996:Solutions; C066696:ncd protein, Drosophila; D000244:Adenosine Diphosphate; D016547:Kinesins