The lipocalin superfamily of proteins functions in the binding
and transport of a variety of important hydrophobic molecules.
Tear lipocalin is a promiscuous lipid binding member of
the family and serves as a paradigm to study the molecular
determinants of ligand binding. Conserved regions in the
lipocalins, such as the G strand and the F-G loop, may
play an important role in ligand binding and delivery.
We studied structural changes in the G strand of holo-
and apo-tear lipocalin using spectroscopic methods including
circular dichroism analysis and site-directed tryptophan
fluorescence. Apo-tear lipocalin shows the same general
structural characteristics as holo-tear lipocalin including
alternating periodicity of a β-strand, orientation
of amino acid residues 105, 103, 101, and 99 facing the
cavity, and progressive depth in the cavity from residues
105 to 99. For amino acid residues facing the internal
aspect of cavity, the presence of a ligand is associated
with blue shifted spectra. The collisional rate constants
indicate that these residues are not less exposed to solvent
in holo-tear lipocalin than in apo-tear lipocalin. Rather
the spectral blue shifts may be accounted for by a ligand
induced rigidity in holo-TL.
Amino acid residues 94 and 95 are consistent with positions
in the F–G loop and show greater exposure to solvent
in the holo- than the apo-proteins. These findings are
consistent with the general hypothesis that the F–G
loop in the holo-proteins of the lipocalin family is available
for receptor interactions and delivery of ligands to specific
Site-directed tryptophan fluorescence was used in combination
with a nitroxide spin labeled fatty acid analog to elucidate
dynamic ligand interactions with specific amino acid residues.
Collisional quenching constants of the nitroxide spin label
provide evidence that at least three amino acids of the
G strand residues interact with the ligand. Stern–Volmer
plots are inconsistent with a ligand that is held in a
static position in the calyx, but rather suggest that the
ligand is in motion. The combination of site-directed tryptophan
fluorescence with quenching by nitroxide labeled species
has broad applicability in probing specific interactions
in the solution structure of proteins and provides dynamic
information that is not attainable by X-ray crystallography.