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

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 °C, corresponding to a free energy of activation, ΔG‡, 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 U3Cd 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. © 1990, American Chemical Society. All rights reserved.