Ligand-linked stability of mutants of the C-domain of calmodulin.
There is a necessary energetic linkage between ligand binding and stability in biological molecules. The critical glutamate in Site 4 was mutated to create two mutants of the C-domain of calmodulin yielding E140D and E140Q. These proteins were stably folded in the absence of calcium, but had dramatically impaired binding of calcium. We determined the stability of the mutant proteins in the absence and presence of calcium using urea-induced unfolding monitored by circular dichroism (CD) spectroscopy. These calcium-dependent unfolding curves were fit to models that allowed for linkage of stability to binding of a single calcium ion to the native and unfolded states. Simultaneous analysis of the unfolding profiles for each mutant yielded estimates for calcium-binding constants that were consistent with results from direct titrations monitored by fluorescence. Binding to the unfolded state was not an important energetic contributor to the ligand-linked stability of these mutants.
Hobson
Kenosha F
KF
University of Mississippi, Dept. of Chemistry and Biochemistry, University, MS 38677, USA.
Housley
Nicole A
NA
Pedigo
Susan
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D008024:Ligands; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry / Q000494:pharmacology
D002147:Calmodulin; D008024:Ligands; D014508:Urea; D002118:Calcium