Proline isomerism leads to multiple folded conformations of calbindin D9k: direct evidence from two-dimensional 1H NMR spectroscopy.
A complete analysis of calbindin D9k by two-dimensional 1H nuclear magnetic resonance spectroscopy has established the existence of two conformations for the folded protein in solution. Well-resolved major and minor resonances in a ratio of 3:1 are observed throughout the 1H NMR spectrum. Two-dimensional exchange experiments show that the major and minor species are related by an equilibrium process. Analysis of short proton-proton distances along the peptide backbone, identified by two-dimensional nuclear Overhauser effect spectroscopy, provides unambiguous evidence that the two forms of the folded protein differ only in the isomerization state of the peptide bond between Gly-42 and Pro-43. Cis-trans isomerism of Pro-43 is thereby directly identified as the cause of multiple conformations for the folded protein in solution. In addition, when Pro-43 is mutated to a glycine residue there is no indication of multiple conformations. These results provide evidence for the possibility of conformational heterogeneity in the native state of globular proteins.
Chazin
W J
WJ
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Kördel
J
J
Drakenberg
T
T
Thulin
E
E
Brodin
P
P
Grundström
T
T
Forsén
S
S
D064026:Calbindins; D006859:Hydrogen; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011392:Proline; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G
D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D006859:Hydrogen; D011392:Proline