Fast Pressure Jumps Can Perturb Calcium and Magnesium Binding to Troponin C F29W

• Published in: Biochemistry (Easton) Vol. 47; no. 46; pp. 12146 - 12158
• Main Authors: Pearson, David S, Swartz, Darl R, Geeves, Michael A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18-11-2008
• Subjects: Amino Acid Substitution; Animals; Binding Sites - genetics; Calcium - chemistry; Calcium - metabolism; Chickens; Magnesium - chemistry; Magnesium - metabolism; Models, Chemical; Muscle, Skeletal - chemistry; Muscle, Skeletal - metabolism; Mutation, Missense; Pressure; Protein Binding - genetics; Spectrometry, Fluorescence - methods; Troponin C - chemistry; Troponin C - genetics; Troponin C - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi801150w

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 μM, 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 μM (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.