HYDROPHOBIC CORE SUBSTITUTIONS IN CALBINDIN D-9K - EFFECTS ON STABILITY AND STRUCTURE

The effects of hydrophobic core mutations on the stability and structu re of the four-helix calcium-binding protein, calbindin D-9k, have bee n investigated. Eleven mutations involving: eight residues distributed within the hydrophobic core of calbindin D-9k were examined. Stabilit ies were measured by denaturant and thermal induced unfolding monitore d by circular dichroism spectroscopy. The mutations were found to exer t large effects on the stability with midpoints in the urea induced un folding varying from 1.8 M for Leu23 --> Gly up to 6.6 M for Va170 --> Leu and free energies of unfolding in the absence of denaturant rangi ng from 6.6 to 27.4 kJ/mol for the Phe66 --> Ala mutant and the wild-t ype, respectively. A significant correlation was found between the dif ference in free energy of unfolding (Delta Delta G(NU)) and the change in the surface area of the side chain caused by the mutation, in agre ement with other studies. Notably, both increases and decreases in sid e-chain surface area caused quantitatively equivalent effects on the s tability. In other words, a correlation between the absolute value of the change in the surface of the side chain and Delta Delta G(NU) was observed with a value of approximately 0.14 kJ M-1 Angstrom(-2). The g enerality of this observation is discussed. Significant effects on the cooperativity of the unfolding reaction were also observed. However, a correlation between the cooperativity and Delta Delta G(NU), which h as been reported in other systems as an indication of effects of mutat ions on the unfolded state, was not observed for calbindin D9k Despite the large effects on Delta Delta G(NU) and cooperativity, the structu res of the mutants in the native form remained intact as indicated by circular dichroism, NMR, and fluorescence measurements. The structural response to calcium-binding was also conserved. The following paper i n this issue [Kragelund, B. B., et al, (1998) Biochemistry 37, 8926-89 37] examines the effects of these mutations on the calcium binding pro perties of calbindin D-9k.