Reaction Kinetics

We use and develop rapid-reaction kinetic equipment to measure protein-ligand interactions. See Molecular Enzymology Group . We mainly use fluorescence detection because of its excellent sensitivity. Probes we exploit include tryptophan, pyrene, GFP and a variety of fluorescent nucleotide analogues. We have developed a detailed reaction mechanism for the Dictyostelium myosin II  ATPase

 

kinetic scheme and record

This stopped-flow record shows a transient rapid decrease in tryptophan fluorescence on mixing Dictyostelium myosin II motor domain with ATP due to M+ATP + M*ATP formation (M+ATP more significant at lower temperatures), followed by a rise in fluorescence as the M*ATP <--> M.*ADP.Pi states requilibrate on hydrolysis of ATP. Relaxation methods (temperature and pressure jump) were required to resolve the M+ATP <--> M*ATP interconversion (which corresponds to switch 2 closing).

We have also applied transient kinetic methods to elucidate the mechanism of binding between other cytokskeletal proteins including actin, talin, a-actinin, vinculin and regulatory proteins such as S100A4. These studies have been carried out in collaboration with structural biologists who have determined the high-resolution structures by X-ray crystallography or NMR spectroscopy:

1lvk X-RAY CRYSTAL STRUCTURE OF THE Mg 2'(3')-O-(N-METHYLANTHRANILOYL) NUCLEOTIDE BOUND TO DICTYOSTELIUM DISCOIDEUM MYOSIN MOTOR DOMAIN

1qkr CRYSTAL STRUCTURE OF THE VINCULIN TAIL AND A PATHWAY FOR ACTIVATION

2k00 Solution structure of the talin F3 in complex with layilin cytodomain

1c1v The 1.5 A Crystal structure of Ca2+-bound S100A4

3MYH 3MYK 3MYL Ser236Ala mutant of Dictyostelium myosin II motor domain

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Department of Molecular and Cell Biology

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E: MolCellBiol@le.ac.uk

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