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.