Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k.
The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.
Wendt
B
B
Physical Chemistry 2, Lund University, Sweden.
Hofmann
T
T
Martin
S R
SR
Bayley
P
P
Brodin
P
P
Grundström
T
T
Thulin
E
E
Linse
S
S
Forsén
S
S
D000596:Amino Acids / Q000032:analysis / Q000502:physiology*; D001059:Apoproteins; D064026:Calbindins; D002135:Calcium-Binding Proteins / Q000032:analysis; D002942:Circular Dichroism; D004355:Drug Stability; D004735:Energy Transfer; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D011489:Protein Denaturation / Q000187:drug effects*; D064030:S100 Calcium Binding Protein G / Q000032:analysis* / Q000235:genetics; D014443:Tyrosine; D014508:Urea / Q000494:pharmacology*
D000596:Amino Acids; D001059:Apoproteins; D064026:Calbindins; D002135:Calcium-Binding Proteins; D064030:S100 Calcium Binding Protein G; D014443:Tyrosine; D014508:Urea