Functional methionines in the collagen/gelatin binding domain of plasma fibronectin: effects of chemical modification by chloramine T.
Chemical modification of plasma fibronectin (pFn) or its 40-kDa collagen/gelatin binding (CGB) domain by low concentrations of chloramine T (CT), a methionine-specific oxidant, caused decreased binding affinity between pFn or the isolated CGB domain and Sepharose-immobilized denatured collagen or a Texas Red-labeled CNBr fragment CB7 from the alpha 1 chain of type I collagen. Kds obtained by fluid-phase fluorescence polarization binding assays increased upon oxidation about 17-fold for pFn and by 4-fold for the CGB domain. Comparison of CT-oxidized and native CGB domains by endogenous tryptophan fluorescence and CD spectra gave no indication of conformational changes. delta GH2O, the free energy of unfolding at infinite denaturant dilution, derived from guanidinium chloride denaturation curves, differed by less than 0.7 kcal/mol for the oxidized and native CGB domains, indicating essentially equivalent conformational stabilities. We show here that methionyl residues found at positions 412, 432, and 446 are the sites of the oxidative modification. Modification protection experiments carried out in the presence of gelatin demonstrated specific protection of otherwise oxidizable methionyl residues and preservation of high-affinity binding. These results implicate methionyl residues as functional in contributing to high-affinity binding interaction between fibronectin and gelatin.
Miles
A M
AM
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130.
Smith
R L
RL
D000595:Amino Acid Sequence; D001665:Binding Sites; D002700:Chloramines / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism*; D003488:Cyanogen Bromide; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014105:Tosyl Compounds
D002700:Chloramines; D005353:Fibronectins; D006146:Guanidines; D016877:Oxidants; D010446:Peptide Fragments; D014105:Tosyl Compounds; C016300:chloramine-T; D005780:Gelatin; D003094:Collagen; D008715:Methionine; D019791:Guanidine; D003488:Cyanogen Bromide