Side chain mobility and ligand interactions of the G strand of tear lipocalins by site-directed spin labeling

Side chain mobility, accessibility, and backbone motion were studied by sit e-directed spin labeling of sequential cysteine mutants of the G strand in tear lipocalins (TL). A nitroxide scan between residues 98 and 105 revealed the alternating periodicity of mobility and accessibility to NiEDDA and ox ygen, characteristic of a beta-strand. Residue 99 was the most inaccessible to NiEDDA and oxygen. EPR spectra with the fast relaxing agent, K3Fe(CN)(6 ), exhibited two nitroxide populations for most residues. The motionally co nstrained population was relatively less accessible to K3Fe(CN)(6) because of dynamic tertiary contact, probably with side chain residues of adjacent strands. With increasing concentrations of sucrose, the spectral contributi on of the immobile component was greater, indicating a larger population wi th tertiary contact. Increased concentrations of sucrose also resulted in a restriction of mobility of spin-labeled fatty acids which were bound withi n the TL cavity. The data suggest that sucrose enhanced ligand affinity by slowing the backbone motion of the lipocalin. The correlation time of an MT SL derivative (I) attached to F99C resulted in the lack of side chain motio n and therefore reflects the overall rotation of the TL complex. The correl ation time of F99C in tears (13.5 ns) was the same as that in buffer and in dicates that TL exists as a dimer under native conditions. TL-spin-labeled ligand complexes have a shorter correlation time than the protein alone, in dicating that the fatty acids are not rigidly anchored in the cavity, but m ove within the pocket. This segmental motion of the ligand was modulated by protein backbone fluctuations. Accessibility studies with oxygen and NiEDD A were performed to determine the orientation and depth of a series of fatt y acid derivatives in the cavity of TL. Fatty acids are oriented with the h ydrocarbon tail buried in the cavity and the carboxyl group oriented toward the mouth. In general, the mobility of the nitroxide varied according to p osition such that nitroxides near the mouth had greater mobility than those located deep in the cavity. Nitroxides positioned up to 16 carbon units fr om the hydrocarbon tail of the ligand are motionally restricted and inacces sible, indicating the cavity extends to at least this depth. EPR spectra ob tained with and without sucrose showed that the intracavitary position of l auric acid in TL is similar to that in P-lactoglobulin. However, unlike bet a-lactoglobulin, TL binds 16-doxyl stearic acid, suggesting less steric hin drance and greater promiscuity for TL.