ATP hydrolysis in human Eg5 kinesin monitored by millisecond time‐resolved FTIR: real‐time dynamics of conformation and chemistry in vitro

Abstract

Human Eg5 (HsEg5), a kinesin motor protein, is capable of ATP hydrolysis and utilizes the energy to move along the microtubule and participate in the spindle pole segregation during mitosis in eukaryotic cells. Herein, we present time‐lapsed monitoring of an in vitro ATP hydrolysis reaction by HsEg5 with ms time resolution. To survey dynamic structural and chemical changes during ATP hydrolysis concomitantly, difference Fourier‐transform infrared spectroscopy was used to monitor HsEg5 catalysis, by triggering initiation of the reaction by UV‐photolysis of caged ATP. A HPO3− vibrational mode was observed in our time‐lapse infrared data at 10 s after ATP release. This mode is assigned to formation of Pi by HsEg5, consistent with steady‐state measurement of enzyme kinetics (Vmax = 0.11 s−1). In the carbonyl stretching, or amide I, vibration of the peptide backbone in the same time‐lapse spectra, four modes were observed, reflecting net changes that occur in the secondary structure of Eg5 in a temporal sequence. These amide I modes indicate four unique conformational changes, or 'switches,' were detected in HsEg5 catalysis that may correspond to different intermediates, such as ATP binding, isomerization, and bond cleavage. Moreover, there is a subset of conformational changes that precede Pi release and there is an independent set of conformational changes as occur in tandem with Pi release.