Millisecond Time-lapsed Monitoring of ATP Hydrolysis by Human Eg5 Kinesin: Real-time Dynamics of Conformation and Chemistry in vitro

Abstract

The hydrolysis of ATP is one of the key chemical reactions in life. Its mechanistic details in biological systems have been a challenge to unravel, as chemical changes in proteins during ATP hydrolysis go hand-in-hand with a series of large-scale conformational alterations. Monitoring the dynamics of chemistry and structure has been experimentally intractable, and requires application of novel methods. Herein, we present time-lapsed monitoring of an in vitro ATP hydrolysis reaction by a kinesin motor protein with 180-millisecond time resolution. Kinesin proteins are one of three major categories of motor proteins, capable of using ATP hydrolysis to power force generation and subsequent movement along cytoskeletal elements in cells. Our model protein system is human Eg5 (HsEg5), a Kinesin-5 motor protein participating in the spindle pole segregation during mitosis in higher eukaryotes. Truncated to its monomeric motor domain, we purified active HsEg5 and confirmed its ability to hydrolyze ATP. To monitor dynamic structural and chemical changes during ATP hydrolysis concomitantly, we used difference Fourier-transform infrared (FTIR) spectroscopy with HsEg5 kinesin samples, triggering initiation of the ATPase reaction by UV-photolysis of caged ATP. Interpretation of these biological data was guided by model compound data on ATP derivatives. The time-lapse data highlighted resolution of two distinct sets of conformational changes: a series of HsEg5 structural changes that precedes ATP hydrolysis and a set of structural alterations that occurs upon onset of ATP hydrolysis. Thus, we conclude that we have the first direct observation of dynamic conformational changes caused by the ATP binding in any kinesin motor protein. Secondly, the structural modifications that occur HsEg5 when ATP hydrolysis is initiated are different than those in the substrate-binding step.