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Moffett, Marque Lou

Biochemistry

2010

MS

The groundbreaking discovery of kinesin in 1985 was largely due to the development of an important video-enhanced microscopy technique some four year earlier. In the twenty-five years since kinesin's discovery, the importance of the enzyme has become increasingly clear. Now known to play biological roles as diverse as intracellular-transportation, spindle pole organization and microtubule depolymerization in organisms of every biological kingdom, researchers around the world are working diligently to understand kinesins from the atomic to the macroscopic levels. Great strides have been made to understand the roles of kinesins in cells. However, due to limitations ofcurrent technologies, we have only very recently begun uncovering thefundamental relationships between the structure and biochemical functions of the enzyme at the atomic level.
The very small conformational changes that are expected to occur within the catalytic site of kinesin continue to escape detection, as techniques such as nuclear magnetic resonance, x-ray crystallography, cryoelectron microscopy, electron paramagnetic resonance and fluorescence spectroscopy do not present the sensitivity required to monitor such changes on the small scale upon which they are expected to occur in kinesins. Presented here is the work undertaken to overcome this small-scale limitation. The use of bimane as an intrinsic fluorescent probe provides the ability to measure conformational changes that occur within the 5 to 20 Å range as they occur in proteins, thereby extending the molecular ruler to include important enzymatic movements that were previously below detection limits.