Disulfide bonds in homo‐ and heterodimers of EF‐hand subdomains of calbindin D_9k: Stability, calcium binding, and NMR studies

Abstract

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.

A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies

“…The method for the measurement of calcium-binding by competitive UV-spectrophotometry can be found in the references from which several of the calcium-binding results are taken (Linse et ai., 1991;Linse et al, 1993). The results presented here and quoted from other studies are the average of several separate determinations.…”

Quantitative measurements of the cooperativity in an EF‐hand protein with sequential calcium binding

“…Determinations of calcium binding constants from titrations in the presence of a chromophoric chelator were made as described previously (Linse et al, , 1993.…”

Human S100b protein: Formation of a tetramer from synthetic calcium‐binding site peptides

“…that observed in other calcium-binding peptides (Kay et al, 1991;Monera et al, 1992;Linse et al, 1993;Shaw et al, 1994). In Figure 4A the aromatic region of 0.1 mM hslbl is shown as a function of temperatures ranging from 10 to 75 "C. At the lower temperatures, the peaks were all broad and featureless, similar to those shown for the initial spectra in the concentrationdependence studies (Fig.…”

“…It has further been determined that synthetic helix-loop-helix peptides can assemble to form two-site homo-and hetero-site domains similar in structure to the native domain found in proteins such as troponin-C and calbindin D9, (Shaw et al, 1990(Shaw et al, , 1992a(Shaw et al, , 1992bKay et al, 1991;Linse et al, 1993). In the present studies we have used this peptide approach to determine if the calcium-binding sites in the dimeric calcium-binding protein SlOOb are organized in a similar pairwise fashion to those in troponin-C, calmodulin, or calbindin D9,, or if the sites in SlOOb adopt a unique arrangement.…”

“…A novel aspect of this approach is that two-site calcium-binding domains can be assembled from a pair of synthetic peptides, each comprised of a single helix-loop-helix sequence and has allowed synthetic twosite domains to be synthesized from peptides containing sites 111 and IV from troponin-C (Shaw et al, 1990(Shaw et al, , 1992a(Shaw et al, , 1992bKay et al, 1991) and sites I and I1 from calbindin Dgk (Finn et al, 1992;Linse et al, 1993). Using high-resolution NMR spectroscopy, it has further been shown that the three-dimensional structures for the two TnC homodimers (Kay et al, 1991;Shaw et al, 1992aShaw et al, , 1992b are symmetrically arranged in a head-to-tail fashion similar in structure to that observed for sites 111 and IV in the X-ray structures of TnC (Herzberg & James, 1988;Satyshur et al, 1988).…”