Metal and substrate binding to an Fe(II) dioxygenase resolved by UV spectroscopy with global regression analysis.
The addition of divalent metal ions or substrate taurine to TauD, an alpha-ketoglutarate-dependent dioxygenase, alters its UV absorption, as clearly observed by monitoring the protein's difference spectra. Binding of metal ions leads to a decrease in absorption at approximately 297 nm and modulation of other features. A separate signature with enhanced absorption at approximately 295 nm is identified for binding of taurine. These narrow ( approximately 700 cm(-1)) and intense ( approximately 0.5mM(-1) cm(-1)) spectral changes are attributed to ligand-induced protein conformational changes affecting the environment of aromatic residues. The changes in the UV difference spectra were exploited to assess directly the thermodynamics and kinetics of ligand interactions in wild-type TauD and selected variants. This approach holds promise as a new tool to probe ligand-induced conformational changes in a wide range of other proteins. Experimental and quantification approaches for a reliable analysis of protein absorption below 320 nm are presented.
Grzyska
Piotr K
PK
Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Hausinger
Robert P
RP
Proshlyakov
Denis A
DA
D019943:Amino Acid Substitution; D007656:Ketoglutaric Acids / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008670:Metals / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012044:Regression Analysis; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013379:Substrate Specificity; D013654:Taurine / Q000737:chemistry; D013816:Thermodynamics
D007656:Ketoglutaric Acids; D008024:Ligands; D008670:Metals; D011994:Recombinant Proteins; D013654:Taurine; D006899:Mixed Function Oxygenases; C433064:taurine-alpha-ketoglutarate dioxygenase