Kinetic Analysis of the Interactions between Troponin C and the C-terminal Troponin I Regulatory Region and Validation of a New Peptide Delivery/Capture System used for Surface Plasmon Resonance

Using surface plasmon resonance (SPR)-based biosensor analysis and fluorescence spectroscopy, the apparent kinetic constants, kon and koff, and equilibrium dissociation constant, Kd, have been determined for the binding interaction between rabbit skeletal troponin C (TnC) and rabbit skeletal troponin I (TnI) regulatory region peptides: TnI96–115, TnI96–131 and TnI96–139. To carry out SPR analysis, a new peptide delivery/capture system was utilized in which the TnI peptides were conjugated to the E-coil strand of a de novo designed heterodimeric coiled-coil domain. The TnI peptide conjugates were then captured via dimerization to the opposite strand (K-coil), which was immobilized on the biosensor surface. TnC was then injected over the biosensor surface for quantitative binding analysis. For fluorescence spectroscopy analysis, the environmentally sensitive fluoroprobe 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (1,5-IAEDANS) was covalently linked to Cys98 of TnC and free TnI peptides were added. SPR analysis yielded equilibrium dissociation constants for TnC (plus Ca2+) binding to the C-terminal TnI regulatory peptides TnI96–131 and TnI96–139 of 89 nM and 58 nM, respectively. The apparent association and dissociation rate constants for each interaction were kon=2.3×105 M−1 s−1, 2.0×105 M−1 s−1 and koff=2.0×10−2 s−1, 1.2×10−2 s−1 for TnI96–131 and TnI96–139 peptides, respectively. These results were consistent with those obtained by fluorescence spectroscopy analysis: Kd being equal to 130 nM and 56 nM for TnC–TnI96–131 and TnC–TnI96–139, respectively. Interestingly, although the inhibitory region peptide (TnI96–115) was observed to bind with an affinity similar to that of TnI96–131 by fluorescence analysis (Kd=380 nM), its binding was not detected by SPR. Subsequent investigations examining salt effects suggested that the binding mechanism for the inhibitory region peptide is best characterized by an electrostatically driven fast on-rate (∼1×108 to 1×109 M−1 s−1) and a fast off-rate (∼1×102 s−1). Taken together, the determination of these kinetic rate constants permits a clearer view of the interactions between the TnC and TnI proteins of the troponin complex.