Electron paramagnetic resonance spectroscopy of nitroxide-labeled calmodulin.
Calmodulin (CaM) is a highly conserved calcium-binding protein consisting of two homologous domains, each of which contains two EF-hands, that is known to bind well over 300 proteins and peptides. In most cases the (Ca(2+))(4-)form of CaM leads to the activation of a key regulatory enzyme or protein in a myriad of biological processes. Using the nitroxide spin-labeling reagent, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl, bovine brain CaM was modified at 2-3 methionines with retention of activity as judged by the activation of cyclic nucleotide phosphodiesterase. X-band electron paramagnetic resonance (EPR) spectroscopy was used to measure the spectral changes upon addition of Ca(2+) to the apo-form of spin-labeled protein. A significant loss of spectral intensity, arising primarily from reductions in the heights of the low, intermediate, and high field peaks, accompanied Ca(2+) binding. The midpoint of the Ca(2+)-mediated transition determined by EPR occurred at a higher Ca(2+) concentration than that measured with circular dichroic spectroscopy and enzyme activation. Recent data have indicated that the transition from the apo-state of CaM to the fully saturated form, [(Ca(2+))(4-)CaM], contains a compact intermediate corresponding to [(Ca(2+))(2-)CaM], and the present results suggest that the spin probes are reporting on Ca(2+) binding to the last two sites in the N-terminal domain, i.e. for the [(Ca(2+))(2)-CaM] → [(Ca(2+))(4-)CaM] transition in which the compact structure becomes more extended. EPR of CaM, spin-labeled at methionines, offers a different approach for studying Ca(2+)-mediated conformational changes and may emerge as a useful technique for monitoring interactions with target proteins.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
Paula B
PB
Bowman
David
D
Puett
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D001923:Brain Chemistry; D002147:Calmodulin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D009589:Nitrogen Oxides / Q000737:chemistry*; D013113:Spin Labels
D002147:Calmodulin; D009589:Nitrogen Oxides; D013113:Spin Labels; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C039900:nitroxyl