Pearson, D.S. and Holtermann, G. and Ellison, P. and Cremo, C. and Geeves, M.A. (2002) A novel pressure-jump apparatus for the microvolume analysis of protein-ligand and protein-protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1. Biochemical Journal, 366 (Part 2). pp. 643-651. ISSN 0264-6021.
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Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50 microl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev. Sci. Instrum. 47, 1383-1393]. This equipment can apply pressure changes of +/-20 MPa (maximally) in time periods as short as 80 micros and follow the resulting change in fluorescence signals. The system is relatively simple to use with fast (approx. 1 min) exchange of samples. In the present study, we show that this system can perturb the binding of 2'(3')-O-(N-methylanthraniloyl)-ADP to myosin subfragment-1(S1) from skeletal and smooth muscles. The kinetic data are consistent with previous work, and in addition show that, although 2'(3')-O-(N-methylanthraniloyl)-ADP binds with a similar affinity to both proteins, the increase in molar volume for the skeletal-muscle S1 binding to ADP is half of that for the smooth-muscle protein. This high-volume change for smooth-muscle S1 may be related to the ability of ADP to induce a 23 degrees tilt in the tail of S1 bound to actin.
|Additional information:||0264-6021 (Print) Journal Article Research Support, Non-U.S. Gov't|
|Uncontrolled keywords:||Adenosine Diphosphate/*metabolism Animals Binding Sites Electrochemistry/instrumentation/methods *Ligands Muscle, Skeletal/*metabolism Muscle, Smooth/*metabolism Myosin Subfragments/*chemistry/*metabolism Pressure|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group|
|Depositing User:||Michael Geeves|
|Date Deposited:||04 Sep 2008 15:40|
|Last Modified:||14 Jan 2010 14:14|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/4011 (The current URI for this page, for reference purposes)|
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