Surface exposure of the methionine side chains of calmodulin in solution -A nitroxide spin label and two-dimensional NMR study

Binding of calcium to calmodulin (CaM) causes a conformational change in th is ubiquitous calcium regulatory protein that allows the activation of many target proteins. Met residues make up a large portion of its hydrophobic t arget binding surfaces. In this work, we have studied the surface exposure of the Met residues in the apo- and calcium-bound states of CaM in solution , Complexes of calcium-CaM with synthetic peptides derived from the CaM-bin ding domains of myosin light chain kinase, constitutive nitric-oxide syntha se, and CaM-dependent protein kinase I were also studied. The surface expos ure was measured by NMR by studying the effects of the soluble nitroxide sp in label, 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy, on the line widt hs and relaxation rates of the Met methyl resonances in samples of biosynth etically C-13-methyl-Met-labeled CaM. The Met residues move from an almost completely buried state in apo-CaM to an essentially fully exposed state in Ca2+, CaM. Binding of two Ca2+ to the C-terminal lobe of CaM causes full e xposure of the C-terminal Met residues and a partial exposure of the N-term inal Met side chains. Binding of the three target peptides blocks the acces s of the nitroxide surface probe to nearly all Met residues, although the m ode of binding is distinct for the three peptides studied. These data show that calcium binding to CaM controls the surface exposure of the Met residu es, thereby providing the switch for target protein binding.