Title
Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca2+-binding affinity in a C-domain mutant of cardiac troponin C
Author
Tiago Veltri, Guilherme A. P. de Oliveira, Ewa A. Bienkiewicz, Fernando L. Palhano, Mayra de A. Marques, Adolfo H. Moraes, Jerson L. Silva, Martha M. Sorenson, Jose R. Pinto
Year
2017
Journal
Scientific Reports
Abstract
The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30–40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.
Full Article
Instrument
FP-8300
Keywords
Fluorescence, Protein structure, Ligand binding, Thermal stability, Biochemistry