Pearson, David S. and Swartz, Darl R. and Geeves, Michael A. (2008) Fast Pressure Jumps Can Perturb Calcium and Magnesium Binding to Troponin C F29W†. Biochemistry, 47 (46). pp. 12146-12158. ISSN 0006-2960.
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We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 muM, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 muM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group|
|Depositing User:||Sue Davies|
|Date Deposited:||09 Oct 2012 13:04|
|Last Modified:||04 Feb 2013 10:38|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/31438 (The current URI for this page, for reference purposes)|
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