DISULFIDE BONDS IN HOMODIMERS AND HETERODIMERS OF EF-HAND SUBDOMAINS OF CALBINDIN-D(9K) - STABILITY, CALCIUM-BINDING, AND NMR-STUDIES

The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39 --> Cys, Ile 73 --> Cys, and Pro 43 --> Met. Backbone H-1 NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by Cd-113 NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases T(m), the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95-degrees-C and C(m), the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bond or the disulfide bond. Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive cooperativity, and an average affinity of 10(6) M-1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment T(m) increases from 59 +/- 2 without covalent linkage to 91 +/- 2-degrees-C with disulfide, and C(m) from almost-equal-to 1.5 to 7.5 M. The overall topology of this homodimer is derived from H-1 NMR assignments and a few key NOEs.