Solid-state NMR measurements of the kinetics of the interaction between phospholamban and Ca2+-ATPase in lipid bilayers.
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 Ca2+-ATPase. In this work solid-state NMR methods have been used to investigate the dynamics of the inhibitory association between PLB and Ca2+-ATPase. Skeletal muscle Ca2+-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 ([alpha-13C-L44]AAA-PLB). In these membranes the PLB variant was found to partially inhibit Ca2+-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 [alpha-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 [alpha-13C-L44]AAA-PLB and Ca2+-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 Ca2+-ATPase was on the order of 2.5 ms at temperatures between 0 degrees C and 30 degrees 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.
Hughes
Eleri
E
Faculty of Life Sciences, University of Manchester, Manchester, UK.
Middleton
David A
DA
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008567:Membranes, Artificial; D008954:Models, Biological; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban