Combining Solution and Solid State NMR Techniques in the Analysis of Arg to Cys Mutations in Phospholamban Associated with Dilated Cardiomyopathy

Abstract

Dilated cardiomyopathy (DCM) is characterized by the enlargement and weakening of the left ventricle. This dilation of the ventricle prevents the heart from contracting normally and can spread to the right ventricle and atria as the disease progresses. Inherited forms of DCM have been identified in families that carry missense, deletion, or nonsense mutations in the regulatory protein, phospholamban (PLN). Two of the identified missense mutations, R9C and R25C, result in cysteine substitutions for arginine residues in PLN's cytoplasmic domain. In vitro studies with these mutants indicate that they have an altered ability to regulate SERCA, however it is unclear how these mutations lead to dysregulation of SERCA.Here we have applied both solution and solid-state NMR techniques to characterize the structure, dynamics and topology of the monomeric constructs for the R9C and R25C mutants to better understand how these mutations affect PLN's regulation of SERCA in the development of DCM. These studies were pursued under both reducing and oxidizing conditions to better reflect the physiological conditions of DCM progression and for comparison to coupled enzyme activity assays used to quantify the effect of these mutants on SERCA activity. We have utilized chemical shift perturbations as sensitive probes toward the local environment of the cytoplasmic and juxta-membrane domains of PLN which harbor the aforementioned mutations. Dynamics in lipid bilayers have been probed through cross-polarization or J-coupling enabled experiments which are exclusively sensitive toward either the rigid or mobile regions of the protein. Heteronuclear NMR was employed to probe the fast timescale dynamics of these mutants. Finally, separated local field experiments in magnetically aligned bicelles were employed to probe the topology.