Constructing a structural model of troponin using site-directed spin labeling: EPR and PRE-NMR
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The relative ease of introducing a paramagnetic species onto a protein, and advances in electron paramagnetic resonance (EPR) over the past two decades, have established spin labeling as a vital structural biology technique for revealing the functional workings of the troponin muscle regulatory complex—an ~80 kDa heterotrimeric protein switch for turning on striated muscle contraction. Through the site-directed spin labeling (SDSL) of cysteine residues at key sites in troponin, a molecular-level understanding of the troponin muscle regulatory system across all levels of structural hierarchy has been achieved. Through the application of EPR, mobility and accessibility trends in the EPR signals of the spin labels attached to consecutive residues can reveal the secondary structure of troponin elements and also help map the interaction between subunits. Distance restraints calculated from the interspin interactions between spin label pairs have helped with building a structural model of the troponin complex. Further, when SDSL is paired with NMR, paramagnetic relaxation enhancement (PRE)-NMR has been used to obtain high-resolution structural detail for both intra- and interdomain interactions in troponin and revealed details of protein conformational changes and dynamics accompanying troponin function. In this review, we provide an overview of the SDSL labeling methodology and its application towards building a dynamic structural model of the multi-subunit troponin complex which details the calcium-induced conformational changes intimately linked to muscle regulation. We also describe how the SDSL method, in conjunction with EPR or NMR, can be used to obtain insights into structural perturbations to troponin caused by disease-causing mutations.
KeywordsCardiac troponin Site-directed spin labeling Electron paramagnetic resonance (EPR) Paramagnetic relaxation enhancement NMR (PRE-NMR) Protein dynamics
EK is supported by a Macquarie University International Post Graduate Research Award. The authors would like to thank Dr. Phani Potluri (Macquarie University) for many helpful discussions.
Compliance with ethical standards
Conflict of interest
Ehsan Kachooei declares that he has no conflict of interest. Nicole M. Cordina declares that she has no conflict of interest. Louise J. Brown declares that she has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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