Metal-induced changes in the fluorescence properties of tyrosine and tryptophan site-specific mutants of oncomodulin.
Oncomodulin is a 108-residue, oncodevelopmental protein containing two calcium-binding sites identified as the CD- and EF-loops. The protein contains no tryptophan and only two tyrosine residues, one which is a calcium ligand in the CD-loop (Tyr-57) and one which lies in the flanking D-helix of this loop (Tyr-65). Site-specific mutagenesis was performed to yield five mutants, two with phenylalanine substituted for tyrosine in positions 57 and 65 and three with tryptophan substituted into position 57 in the CD-loop, position 65 in the D-helix, and position 96 in the EF-loop. The single Tyr-containing mutants demonstrated that position 57 was perturbed to a significantly greater extent than position 65 upon calcium binding. Although both tyrosine residues responded to decalcification, the fluorescence intensity changes were in opposite directions, with the more dominant Tyr-57 accounting for the majority of the intrinsic fluorescence observed in native oncomodulin. The substitution of tryptophan for each tyrosyl residue revealed that in both positions the tryptophan resided in polar, conformationally heterogeneous environments. The environment of Trp-57 was affected by Ca2+ binding to a much greater extent compared to that of Trp-65. Only 1 equiv of Ca2+ was required to produce greater than 70% of the Trp fluorescence changes in positions 57 and 65, indicating that Ca2+ binding to the higher affinity EF-loop had a pronounced effect on the protein structure.(ABSTRACT TRUNCATED AT 250 WORDS)
Hutnik
C M
CM
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada.
MacManus
J P
JP
Banville
D
D
Szabo
A G
AG
D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D013057:Spectrum Analysis; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D002135:Calcium-Binding Proteins; D008670:Metals; D009363:Neoplasm Proteins; C034733:oncomodulin; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium