We have used membrane surface charge to modulate the structural dynamics of an integral membrane protein, phospholamban (PLB), and thereby its functional inhibition of the sarcoplasmic reticulum Ca-ATPase (SERCA). It was previously shown by electron paramagnetic resonance, in vesicles of neutral lipids, that the PLB cytoplasmic domain is in equilibrium between an ordered T state and a dynamically disordered R state and that phosphorylation of PLB increases the R state and relieves SERCA inhibition, suggesting that R is less inhibitory. Here, we sought to control the T/R equilibrium by an alternative means-varying the lipid headgroup charge, thus perturbing the electrostatic interaction of PLB's cationic cytoplasmic domain with the membrane surface. We resolved the T and R states not only by electron paramagnetic resonance in the absence of SERCA but also by time-resolved fluorescence resonance energy transfer from SERCA to PLB, thus probing directly the SERCA-PLB complex. Compared to neutral lipids, anionic lipids increased both the T population and SERCA inhibition, while cationic lipids had the opposite effects. In contrast to conventional models, decreased inhibition was not accompanied by decreased binding. We conclude that PLB binds to SERCA in two distinct structural states of the cytoplasmic domain: an inhibitory T state that interacts strongly with the membrane surface and a less inhibitory R state that interacts more strongly with the anionic SERCA cytoplasmic domain. Modulating membrane surface charge provides an effective way of investigating the correlation between structural dynamics and function of integral membrane proteins.
Bibliographical noteFunding Information:
Fluorescence and EPR experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Computational resources were provided by the Minnesota Supercomputing Institute. We thank Razvan Cornea and Gianluigi Veglia for helpful discussions. Special thanks to Octavian Cornea for assistance with manuscript preparation and submission. This study was supported by National Institutes of Health grants to D.D.T. ( R01 GM27906 and P30 AR0507220 ). J.L. was supported by a University of Minnesota Doctoral Dissertation Fellowship , and Z.M.J. was supported by National Institutes of Health training grant T32 AR007612 .
- electrostatic interactions