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 pro tein rat oncomodulin (OM); F102W, Y57W, Y65W and the engineered protei n CDOM33 which had the 12 residues of the CD loop replaced with a more potent metal binding site. Both the thermal and the chemical stabilit y 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 th ermostable, 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 che mical denaturation, the engineered protein showed the highest stabilit y. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordi nate number (i.e., Tb3+) which binds more carboxylate ligands, or by p roviding more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability. (C) 1998 Elsevier Science B .V. All rights reserved.