title: The rate and structural consequences of proline cis-trans isomerization in calbindin D9k: NMR studies of the minor (cis-Pro43) isoform and the Pro43Gly mutant.
abstract: The EF-hand calcium-binding protein, calbindin D9k, exists in solution in the calcium-loaded state, as a 1:3 equilibrium mixture of two isoforms, the result of cis-trans isomerism at the Gly42-Pro43 peptide bond [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198]. Nuclear magnetic resonance (NMR) studies of the minor (cis-Pro43) isoform and the Pro43----Gly mutant are reported here. The rate of cis----trans isomerization at the Pro43 peptide bond in the wild-type protein was determined by line-shape analysis at elevated temperatures, using a sample in which all amino acids, except Ser and Val, were deuterated. The cis----trans rate is calculated to be 0.2 s-1 at 25 degrees C, corresponding to a free energy of activation, delta G, of 77 kJ/mol. The complete sequence-specific 1H NMR assignments of the cis-Pro43 isoform and the Pro43----Gly mutant in the calcium-loaded state have been obtained by using standard methods combined with comparisons to the previously assigned major (trans-Pro43) isoform. This has permitted detailed comparative analysis of 1H NMR chemical shifts, backbone scalar coupling constants, and nuclear Overhauser effects. The minor isoform has a global fold that is identical with that of the major isoform. Structural changes imposed by cis-trans isomerization at Pro43 are highly localized to the linker loop (containing Pro43) that joins the two EF hands. The Pro43----Gly mutant has a global fold that is identical with the wild-type protein, but does not exhibit conformational heterogeneity. Only very limited structural differences are observed between mutant and wild-type protein, and these are also highly localized to the linker loop. The ion-binding properties of the mutant, as determined by 43Ca and 113Cd NMR, are found to be very similar to the wild-type protein. These results provide crucial evidence that justifies the calculation of high-resolution three-dimensional structures of the Pro43Gly mutant, rather than of the conformationally heterogeneous wild-type protein.
authors.affiliation: Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
authors.forename: J
authors.initials: J
authors.lastname: Kördel
authors.forename: S
authors.initials: S
authors.lastname: Forsén
authors.forename: T
authors.initials: T
authors.lastname: Drakenberg
authors.forename: W J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D000595:Amino Acid Sequence; D064026:Calbindins; D002151:Calorimetry; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*
chemical_list: D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D011392:Proline