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.
Physical Chemistry 2, Chemical Centre, Lund, Sweden.
C
C
Johansson
P
P
Brodin
T
T
Grundström
E
E
Thulin
S
S
Forsén
T
T
Drakenberg
D000409:Alanine; D000818:Animals; D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005998:Glycine; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D011392:Proline; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D013696:Temperature
D064026:Calbindins; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D011392:Proline; D000409:Alanine; D002118:Calcium; D005998:Glycine