Solid-state NMR measurements of the kinetics of the interaction between phospholamban and Ca^2 +-ATPase in lipid bilayers

Abstract

Phospholamban (PLB) is a small transmembrane protein that regulates calcium transport across the sarcoplasmic reticulum (SR) of cardiac cells via a reversible inhibitory interaction with Ca^2 +-ATPase. In this work solid-state NMR methods have been used to investigate the dynamics of the inhibitory association between PLB and Ca^2 +-ATPase. Skeletal muscle Ca^2 +-ATPase was incorporated into phosphatidylcholine membranes together with a ten-fold excess of a null-cysteine mutant of PLB labelled with ¹³C at Leu-44 in the transmembrane domain ([α-¹³C-L44]AAA-PLB). In these membranes the PLB variant was found to partially inhibit Ca^2 +-ATPase by reducing the affinity of the enzyme for calcium. Cross-polarization magic angle spinning (CP-MAS) ¹³C NMR spectra of the membranes exhibited a signature peak from [α-¹³C-L44]AAA-PLB at 56 ppm. Changes in the intensity of the peak were observed at different temperatures, which was diagnostic of direct interaction between [α-¹³C-L44]AAA-PLB and Ca^2 +-ATPase. Measurements of dipolar couplings between the ¹³C label and neighbouring protons were analysed to show that the mean residency time for the association of AAA-PLB with Ca^2 +-ATPase was on the order of 2.5 ms at temperatures between 0°C and 30°C. This new NMR approach will be useful for examining how the association of the two proteins is affected by physiological stimuli such as kinases and the elevation of calcium concentration.

• Magic-angle spinning
• SERCA
• dipolar coupling
• cardiovascular disease