Biophysical studies of engineered mutant proteins based on calbindin D_9k modified in the pseudo EF‐hand

Abstract

The genes for four mutant proteins from calbindin all with mutations in the N-terminal Ca2+-binding domain (pseudo EF-hand) have been synthesized and expressed in Escherichia coli. The purification scheme has been modified to minimize the formation of deamidated proteins. The set of modifications in the pseudo EF-hand is an attempt to turn this site into a structure resembling an archetypal EF-hand, with its characteristic 'I3Cd-NMR shift (-80 to -110 ppm) and high calcium-binding constants, whereas the C-terminal Ca2+-binding site (EF-hand) is kept intact in all mutant proteins. The mutant proteins studied here all have pseudo EF-hands with a lower calcium-binding constant and a higher calcium off-rate to the pseudo EF-hand than the wild-type protein.From the results obtained it is obvious that proline 20 in the pseudo EF-hand, which has been deleted or replaced by glycine in three of the mutants, has a stabilizing effect on calcium binding to that site. Furthermore, the modifications in the pseudo EF-hand seem to have only a local effect, leaving the tertiary structure of the protein and the calcium-binding properties of the unmodified site virtually unchanged.

The Lin12-Notch Repeats of Pregnancy-associated Plasma Protein-A Bind Calcium and Determine Its Proteolytic Specificity

“…Assays in which the effect of Ca 2ϩ was investigated were carried out in distilled (3ϫ) water. Some assays were carried out in the presence of EDTA, EGTA, or calcium-free bovine calbindin D9k (38). As judged by SDS-PAGE and compositional amino acid analysis, the preparation of calbindin was Ͼ95% pure.…”

A paramagnetic probe to localize residues next to carboxylates on protein surfaces

“…Protein expression [31] and purification [32] of both the Ca 2+ and the apo form of bovine calbindin D 9k were performed as reported. The P43M mutant [33,34] was used to avoid any conformational heterogeneity due to cis-trans isomerization as found for the wildtype protein.…”

Characterization of the N‐terminal half‐saturated state of calbindin D9k: NMR studies of the N56A mutant

“…The protein was overexpressed from Escherichia coli strain MM 294 using the expression vector pRCBl (Brodin et al, 1989) and purified as described previously (Johansson et al, 1990). The purified protein was stored at -20 "C as a lyophilized powder containing at least 2.5 molar equivalents of calcium.…”