Calcium-binding properties of cardiac and skeletal troponin C as determined by circular dichroism and ultraviolet difference spectroscopy
Authors: Hincke M. T. , McCubbin W. D. , Kay C. M. 
Published: December 2009

Calcium titration of the conformational change in cardiac and skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2 to 7.4. Computer analysis was used to resolve the contributions from the different classes of Ca2+-binding sites. At pH 6.94 in skeletal TN-C, apparent affinity constants for calcium of 1.8 × 107 and 4.5 × 10s M−1 were determined for the two classes of binding sites. The more sophisticated computer analysis of the data has revealed a substantial CD contribution from the low-affinity sites (~30% of the high affinity contribution at pH 6.94) and suggests that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-affinity sites are occupied, in agreement with a recent nuclear magnetic resonance (NMR) study on this system (Seaman, K. B., Hartshorne, D. J. &Bothner-By, A. A. (1977) Biochemistry 16,4039–4046). With the cardiac protein at pH 7.07, an apparent affinity constant for calcium of 2.0 × 107 M−1 was calculated while no low-affinity site at this pH was detected by CD. On the other hand, at lower pH values, such as 6.05, a CD contribution from the cardiac low-affinity Ca2+-binding site is detected with an apparent binding constant of 3.7 ± 0.7 × 104 M−1.At the lower pH values, protonation of a class of carboxyl groups in each protein which possesses a high pKa (6.2–6.3) elicits the conformational change at the high-affinity sites with a corresponding decrease in the overall magnitude of the Ca2+-evoked changes. The expression of a conformational change upon Ca2+ binding at the level of the low-affinity sites is enhanced by protonation of a class of carboxyls with a pKa of 6.3 in cardiac TN-C and 6.7–6.8 with the skeletal homologue. In both cases, this contribution is reduced upon protonation of carboxyls with pKa ≤ 5.5.It was also observed that the low-affinity sites of skeletal TN-C have a much larger role to play in the total conformational change than the low-affinity sites of cardiac TN-C, a finding probably related to the inability of site 1 in the cardiac protein to bind calcium.In the cardiac protein, the Ca2+-induced tyrosine difference-spectrum maximum is reduced from ΔεM,287 nm = 330 M−1∙cm−1 to 20 M−1∙cm−1 by protonation of a class of groups with a pKa of 6.4, presumably the same carboxyl groups as those involved in the CD conformational contribution from the high-affinity binding sites. No such effect was observed for the skeletal protein where ΔεM,287 nm was constant at 110 M−1∙cm−1 over the pH range studied. The dramatic alterations in the tyrosine environment of cardiac TN-C with pH are attributed to either or both of the tyrosines located in the two high-affinity Ca2+-binding sites (sites 3 and 4). Since skeletal TN-C lacks a tyrosine which is homologous to that found in site 4 of cardiac TN-C and at the same time exhibits a tyrosine difference spectrum that is pH independent, the large effect noted with cardiac TN-C probably originates at the tyrosine in site 4.These results suggest that important classes of carboxyl groups regulate Ca2+ binding and its expression as a conformational change in both skeletal and cardiac troponin C.