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Protein Science, Vol 7, Issue 1 150-157, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

Mapping fatty acid binding to {beta}-lactoglobulin: Ligand binding is restricted by modification of Cys 121

M. NARAYAN and L. J. BERLINER
The Ohio State University Biophysics Program and the Department of Chemistry, The Ohio State University, Columbus, Ohio 43210

Native {beta}-lactoglobulin (Blg) binds 1 mole of palmitic acid per mole of protein with a dissociation constant of 0.6 {mu}M 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 methyl-methanethiosulfonate 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.
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M. Collini, L. D'Alfonso, H. Molinari, L. Ragona, M. Catalano, and G. Baldini
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