BIOPHYSICAL STUDIES OF ENGINEERED MUTANT PROTEINS BASED ON CALBINDIN-D9K MODIFIED IN THE PSEUDO EF-HAND

The genes for four mutant proteins from calbindin D9k, 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 113Cd‐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. Copyright © 1990, Wiley Blackwell. All rights reserved