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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.
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