Effects of metal binding affinity on the chemical and thermal stability of site-directed mutants of rat oncomodulin.
Tryptophan fluorescence was used to study the stability and unfolding behavior of several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM); F102W, Y57W, Y65W and the engineered protein CDOM33 which had the 12 residues of the CD loop replaced with a more potent metal binding site. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca2+ < Tb3+. During thermal denaturation, the transition midpoints (T(un)) of Y65W was the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which acted through the disruption of hydrogen bonds at the C-terminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinate number (i.e., Tb3+) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability.
Zheng
L
L
Department of Chemistry, Brock University, St. Catharines, Ontario, Canada.
Hogue
C W
CW
Brennan
J D
JD
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D006860:Hydrogen Bonding; D008670:Metals / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D013696:Temperature; D013725:Terbium / Q000378:metabolism; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics
D000178:Acrylamides; D002135:Calcium-Binding Proteins; D008670:Metals; D009363:Neoplasm Proteins; C034733:oncomodulin; D013725:Terbium; D020106:Acrylamide; D014364:Tryptophan; D019791:Guanidine; D002118:Calcium