Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-[[2-[bis(carboxymethyl)-amino]- 5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline or by using a Ca2+-selective electrode. The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3. Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1). The binding entropy is strongly positive in all cases. Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants. Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4. These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4). There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.

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Calbindin D,, is a 7kesiduc globular protein made up of two Ca" binding subdomains of the EF-hand type. In order to examine the subdomains independently. a method was devised to selectively cleave the loop between them. Using site-directed mutagenesis, a unique mcthioninc was substituted for Pro"' in the loop. thus nllowiny cleavage usingcyanogen bromide. Agarose gel clectrophoresis shows that the framents have a high affinity for one another, although less so in the absence ofcalcium. 'H-NMR spectra of the fragments indicate that the structures of the heterodimcrs are changed little from that of the intact protein. However, the Ca'+ binding constants of the individual subdomains arc several orders of magnitude lower lhan for lhc corresponding sites in the unclcaved protein.NMR; Site-directed mutagenesis,

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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 Dgk mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39 -+ Cys, Ile 73 -+ Cys, and Pro 43 + Met. Backbone 'H 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 Ii3Cd 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,,,, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 "C and C,,,, 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 IO6 M-I. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment T,,, increases from 59 k 2 without covalent linkage to 91 k 2 "C with disulfide, and C, from = 1.5 to 7.5 M. The overall topology of this homodimer is derived from 'H NMR assignments and a few key NOES.