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+.(ABSTRACT TRUNCATED AT 250 WORDS)