Spin-labeling studies of the conformation of the Ca(2+)-regulatory protein calmodulin in solution and bound to the membrane skeleton in erythrocyte ghosts: implications to transmembrane signaling.
Electron paramagnetic resonance (EPR) studies of the Ca(2+)-regulatory protein calmodulin (CaM) have been performed. The conformation of CaM in solution changes upon binding of Ca2+ allowing the protein to bind to target proteins existing in the red blood cell membrane. In this study a maleimide spin label, covalently attached to the single cysteine residue of CaM located in the first Ca(2+)-binding domain, was used to monitor allosteric conformational changes induced by interaction of CaM with Ca2+ and subsequently with the red blood cell membrane. The results show, relative to apo-CaM, a significant increase in the apparent rotational correlation time, tau, of the spin label when Ca2+ was present in solution (P less than 0.001). When apo-CaM exposed to red blood cell membrane ghosts in the absence of Ca2+, no significant difference in spin label motion was seen relative to solution, consistent with the idea that Ca2+ is required for CaM to bind to skeletal proteins. When Ca2+ was added to CaM which was then exposed to ghosts, a highly significant increase in tau (decrease in motion) (P less than 0.000001) relative to apo-CaM exposed to ghosts was found. This latter increase in tau is significantly greater than that produced by the addition of Ca2+ to CaM in solution (P less than 0.001). The major interaction sites of CaM were found by photoaffinity labeling and autoradiography on SDS-PAGE to be on the principal skeletal protein, spectrin. EPR was also used to investigate the biophysical correlates of transmembrane signaling. Spin-labeled CaM was bound to the membrane skeleton in the presence of Ca2+. On the opposite side of the erythrocyte membrane a lectin was bound to the external glycoconjugate of Band 3, the major transmembrane protein of the erythrocyte. A highly significant increase in T of the maleimide spin probe was found relative to the control system in which the lectin was absent. (P < 0.00001). These results suggest that electron paramagnetic resonance spectra of spin-labeled CaM can provide useful information about protein structure and function when in solution and when bound to membranes.
Yacko
M A
MA
Department of Chemistry, University of Kentucky, Lexington 40506-0055.
Butterfield
D A
DA
D001345:Autoradiography; D001665:Binding Sites; D002147:Calmodulin / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D003208:Concanavalin A; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D008961:Models, Structural; D011487:Protein Conformation; D015398:Signal Transduction; D013113:Spin Labels
D002147:Calmodulin; D003497:Cyclic N-Oxides; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D013113:Spin Labels; D003208:Concanavalin A; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl