Mapping fatty acid binding to beta-lactoglobulin: Ligand binding is restricted by modification of Cys 121.
Native beta-lactoglobulin (Blg) binds 1 mole of palmitic acid per mole of protein with a dissociation constant of 0.6 microM for the primary fatty acid binding site. Chemical modification of Cys 121, which lies at the external putative hydrophobic binding site of Blg, does not affect retinol or 4,4'-bis 1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) binding to the protein, indicating that the incorporated appendages do not perturb the internal hydrophobic site within the beta-barrel of Blg (i.e., the retinoid site is unaffected). On the other hand, methylation of Cys 121, reduces the affinity of Blg for palmitic acid by 10-fold as monitored by intrinsic fluorescence. Modification of the Cys 121 with methylmethanethiosulfonate or a thiol-specific spin label appears to either further weaken or totally eliminate fatty acid binding, respectively, due to steric hindrance. Furthermore, this binding pattern has been independently verified using a spin labeled fatty acid analog and monitoring ESR as well as by bis-ANS fluorescence when bound to the protein. These results suggest that fatty acids bind at the "external site" of beta-lactoglobulin, between the sole alpha-helix and the beta-barrel. In addition, structural stability studies of native and chemically modified Blg appear to confirm this observation as well.
Narayan
M
M
Department of Chemistry, The Ohio State University, Columbus 43210, USA.
Berliner
L J
LJ
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002417:Cattle; D003497:Cyclic N-Oxides / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D008024:Ligands; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D015394:Molecular Structure; D019308:Palmitic Acid / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D013113:Spin Labels; D014801:Vitamin A / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D007782:Lactoglobulins; D008024:Ligands; D013113:Spin Labels; D014801:Vitamin A; C005072:5-doxylstearic acid; D019308:Palmitic Acid; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D008741:Methyl Methanesulfonate; D003545:Cysteine