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 calci um binding properties of calbindin D9k has been addressed in studies o f a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73 C), 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 dis ulfide is inserted on the opposite end of the protein molecule with re spect to the calcium sites, to avoid direct interference with these si tes, as confirmed by Cd-113 NMR. The effect of the disulfide bond on c alcium binding was assessed by titrations in the presence of a chromop horic chelator. A small but significant effect on the cooperativity wa s found, as well as a very modest reduction in calcium affinity. The d isulfide bond increases T(m), the transition midpoint of thermal denat uration, 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. Cal bindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bo nd or the disulfide bond. Kinetic remixing experiments show that separ ated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-ha nd, 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 homo dimer. For the homodimer of the C-terminal fragment T(m) increases fro m 59 +/- 2 without covalent linkage to 91 +/- 2-degrees-C with disulfi de, and C(m) from almost-equal-to 1.5 to 7.5 M. The overall topology o f this homodimer is derived from H-1 NMR assignments and a few key NOE s.