Hajdo Lukasz, Skowronek Krzysztof, Kasprzak Andrzej A
Motor Proteins Laboratory, Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
Arch Biochem Biophys. 2004 Jan 15;421(2):217-26. doi: 10.1016/j.abb.2003.11.009.
Kinesins are molecular motors that produce mechanical work at the expense of ATP hydrolysis. Here, we studied Ncd (non-claret disjunctional), a (-)-end-directed member of this superfamily. To gain insight into the mechanism by which Ncd generates force and movement, we measured distances between the heads in dimeric Ncd-250-700 using fluorescence resonance energy transfer (FRET). About 5% of Ncd heads were labeled with 1,5-IAEDANS (donor), and the remaining thiol groups were modified with QSY35-iodoacetamide (acceptor). Several lines of experimental evidence suggest that the probes were conjugated to Cys-670 in each head of the dimer. The measured donor-acceptor distance was about 35 A. Nucleotides (ADP, ATP, and AMP-PNP) in the presence and absence of microtubules had only small effects on the interhead distances. Similar results were obtained for bidirectional Ncd mutant in which Asn-340 was replaced by a lysine. The results argue against models of Ncd movement in which the heads undergo large spatial rearrangements during mechanochemical cycle and suggest Gly-347 as a possible pivot point for the head rotation.
驱动蛋白是一类分子马达，通过消耗ATP水解产生机械功。在此，我们研究了Ncd（非红化分离），它是这个超家族中一种向（-）端运动的成员。为了深入了解Ncd产生力和运动的机制，我们使用荧光共振能量转移（FRET）测量了二聚体Ncd-250-700中头部之间的距离。约5%的Ncd头部用1,5-IAEDANS（供体）标记，其余巯基用QSY35-碘乙酰胺（受体）修饰。几条实验证据表明，这些探针与二聚体每个头部的Cys-670相连。测得的供体-受体距离约为35埃。在有和没有微管存在的情况下，核苷酸（ADP、ATP和AMP-PNP）对头部间距离只有微小影响。对于将Asn-340替换为赖氨酸的双向Ncd突变体也得到了类似结果。这些结果与Ncd运动模型相悖，在该模型中头部在机械化学循环中经历大的空间重排，并表明Gly-347可能是头部旋转的枢轴点。
