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          Institute: MPI für molekulare Physiologie     Collection: Abteilung III - Physikalische Biochemie     Display Documents

ID: 6089.0, MPI für molekulare Physiologie / Abteilung III - Physikalische Biochemie
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
Authors:Pearson, David S.; Holtermann, Georg; Ellison, Patricia; Cremo, Christine; Geeves, Michael A.
Research Context:protein-protein interactions
Date of Publication (YYYY-MM-DD):2002-09-01
Title of Journal:Biochemical Journal
Journal Abbrev.:Biochem. J.
Start Page:643
End Page:651
Sequence Number of Article:1
Review Status:Peer-review
Audience:Experts Only
Abstract / Description: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 mul) 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 mus 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 (SI) 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 23degrees tilt in the tail of S1 bound to actin.
Free Keywords:ligand binding; molar volume change; pressure relaxation; strain sensing
External Publication Status:published
Document Type:Article
Affiliations:MPI für molekulare Physiologie/Abteilung III - Physikalische Biochemie
External Affiliations:Univ Kent, Dept Biosci, Canterbury CT2 7NJ, Kent, England;
Univ Nevada, Dept Biochem, Reno, NV 89557 USA
Identifiers:URL:http://www.biochemj.org/bj/366/0643/3660643.pdf [pdf version of this article]
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