Single kinesin molecules studied with a molecular force clamp

摘要: Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each its heads alternately sequence. Molecular details how the chemical energy stored ATP coupled to mechanical displacement remain obscure. To shed light on this question, force clamp was constructed, based feedback-driven optical trap capable maintaining constant loads single kinesin motors. The instrument provides unprecedented resolution molecular motion and permits mechanochemical studies under controlled external loads. Analysis records variable concentrations revealed several new features. First, stepping appears be tightly hydrolysis over wide range forces, with per 8-nm advance. Second, stall depends concentration. Third, increased reduce maximum velocity as expected, but also raise apparent Michaelis-Menten constant. cycle therefore contains at least one load-dependent transition affecting rate which molecules bind subsequently commit hydrolysis. It likely other exists, turnover number. Together, these findings will necessitate revisions our understanding motors function.