PNNL

Abstract

We have used frequency-domain fluorescence spectroscopy to investigate the structural linkage between the transmembrane and cytosolic domains of the regulatory protein phospholamban (PLB). Using an engineered PLB having a single cysteine (Cys24) derivatized with the fluorophore N-(1-pyrenyl) maleimide (PMal), we have used fluorescence resonance energy transfer (FRET) to measure the average spatial separation and conformational heterogeneity between PMal bound to Cys24 in the transmembrane domain and Tyr6 in the cytosolic domain near the amino-terminus of PLB. In these measurements, PMal serves as an FRET donor and Tyr6 serves as a FRET acceptor following its nitration by tetranitromethane. The native structure of PLB is retained following site-directed mutagenesis and chemical modification, as indicated by the ability of the derivatized PLB to fully regulate the Ca-ATPase following their co-reconstitution. To assess how phosphorylation modulates the structure of PLB, FRET measurements were made following reconstitution of PLB in membrane vesicles made from lipids extracted from sarcoplasmic reticulum vesicles in the absence of the Ca-ATPase. We find that the cytosolic domain of PLB assumes a wide range of structures relative to the transmembrane sequence, consistent with other structural data indicating the presence of a flexible hinge region between the transmembrane and cytosolic domains of PLB. Phosphorylation of Ser16 by PKA results in almost a two-fold decrease in conformational heterogeneity, suggesting a stabilization of the hinge region of PLB possibly through an electrostatic-linkage between phosphoserine16 and Arg13. These results suggest that the stabilization of the structure of PLB following phosphorylation of Ser16 is part of a switching mechanism, which functions to alter binding interactions between PLB and the nucleotide-binding domain of the Ca-ATPase that modulates enzyme inhibition.