A CONSERVED DISULFIDE MOTIF IN HUMAN TEAR LIPOCALINS INFLUENCES LIGAND-BINDING

Structural and functional characteristics of the disulfide motif have been determined for tear lipocalins, members of a novel group of prote ins that carry lipids, Amino acid sequences for two of the six isolate d isoforms were assigned by a comparison of molecular mass measurement s with masses calculated from the cDNA-predicted protein sequence and available N-terminal protein sequence data. A third isoform was tentat ively sequence assigned using the same criteria. The most abundant iso form has a measured mass of 17 446.3 Da, consistent with residues 19-1 76 of the putative precursor (calculated mass 17 445.8 Da). Chemical d erivatization of native and reduced/denatured protein confirmed the pr esence of a single intramolecular disulfide bond in the native protein , Reactivity of native, reduced, and denatured protein with 4-pyridine disulfide and dithiobis(2-nitrobenzoic acid) indicated that access to the free cysteine is markedly restricted by the intact disulfide brid ge. Mass measurements of tryptic fragments identified C-119 as the fre e cysteine and showed that the single intramolecular disulfide bond jo ined residues C-79 and C-171 Circular dichroism indicated that tear li pocalins have a predominant beta-pleated sheer structure (44%) that is essentially retained after reduction of the disulfide bond, Circular dichroism in the far-UV showed reduced molecular asymmetry and enhance d urea-induced unfolding with disulfide reduction indicative of relaxa tion of protein structure. Circular dichroism in the near-UV shows tha t the disulfide bond contributes to the asymmetry of aromatic sites. T he effect of disulfide reduction on ligand binding was monitored using the intrinsic optical activity of bound retinol. The intact disulfide bond diminishes the affinity of tear lipocalins for retinol and restr icts the displacement of native lipids by retinol. Disulfide reduction is accompanied by a dramatic alteration in ligand-induced conformatio nal changes that involves aromatic residues. The disulfide bridge in t ear lipocalins is important in conferring protein rigidity and influen cing ligand affinity. The disulfide bond appears highly conserved so t hat these findings may have implications for the entire lipocalin supe rfamily.