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Troponin is the Ca 2+ molecular switch that regulates striated muscle contraction. In the heart, troponin Ca 2+ sensitivity is also modulated by the PKA-dependent phosphorylation of a unique 31-residue N-terminal extension region of the Troponin I subunit (NH 2 -TnI). However, the detailed mechanism for the propagation of the phosphorylation signal through Tn, which results in the enhancement of the myocardial relaxation rate, is difficult to examine within whole Tn. Several models exist for how phosphorylation modulates the troponin response in cardiac cells but these are mostly built from peptide-NMR studies and molecular dynamics simulations. Here we used a paramagnetic spin labeling approach to position and track the movement of the NH 2 -TnI region within whole Tn. Through paramagnetic relaxation enhancement (PRE)-NMR experiments, we show that the NH 2 -TnI region interacts with a broad surface area on the N-domain of the Troponin C subunit. This region includes the Ca 2+ regulatory Site II and the TnI switch-binding site. Phosphorylation of the NH 2 -TnI both weakens and shifts this region to an adjacent site on TnC. Interspin EPR distances between NH 2 -TnI and TnC further reveal a phosphorylation induced re-orientation of the TnC N-domain under saturating Ca 2+ conditions. We propose an allosteric model where phosphorylation triggered cooperative changes in both the interaction of the NH 2 -TnI region with TnC, and the re-orientation of the TnC interdomain orientation, together promote the release of the TnI switch-peptide. Enhancement of the myocardial relaxation rate then occurs. Knowledge of this unique role of phosphorylation in whole Tn is important for understanding pathological processes affecting the heart., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

The absence of a crystal structure of the calcium free state of the cardiac isoform of the troponin complex has hindered our understanding of how the simple binding of Ca 2+ triggers conformational changes in troponin which are then propagated to enable muscle contraction. Here we have used continuous wave (CW) and Double Electron-Electron Resonance (DEER) pulsed EPR spectroscopy to measure distances between TnI and TnC to track the movement of the functionally important regulatory 'switch' region of cardiac Tn. Spin labels were placed on the switch region of Troponin I and distances measured to Troponin C. Under conditions of high Ca 2+ , the interspin distances for one set (TnI151/TnC84) were 'short' (9-10Å) with narrow distance distribution widths (3-8Å) indicating the close interaction of the switch region with the N-lobe of TnC. Additional spin populations representative of longer interspin distances were detected by DEER. These longer distance populations, which were ∼16-19Å longer than the short distance populations, possessed notably broader distance distribution widths (14-29Å). Upon Ca 2+ removal, the interspin population shifted toward the longer distances, indicating the release of the switch region from TnC and an overall increase in disorder for this region. Together, our results suggest that under conditions of low Ca 2+ , the close proximity of the TnI switch region to TnC in the cardiac isoform is necessary for promoting the interaction between the regulatory switch helix with the N-lobe of cardiac Troponin C, which, unlike the skeletal isoform, is largely in a closed conformation., (Copyright © 2017 Elsevier Inc. All rights reserved.)