Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.
The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.
Skelton
N J
NJ
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
Kördel
J
J
Akke
M
M
Chazin
W J
WJ
D000577:Amides / Q000737:chemistry*; D002104:Cadmium / Q000737:chemistry* / Q000378:metabolism; D064026:Calbindins; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002614:Chelating Agents; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011522:Protons; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents; D014961:X-Ray Diffraction
D000577:Amides; D064026:Calbindins; D002614:Chelating Agents; D011522:Protons; D064030:S100 Calcium Binding Protein G; D012997:Solvents; D002104:Cadmium; D002118:Calcium