Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: Evidence for features that confer promiscuity in ligand binding

The solution structure of human TL was deduced from the position of the emi ssion peaks after site-directed tryptophan fluorescence (SDTF). The fluores cent amino acid tryptopban was sequentially substituted for each native ami no acid in the sequence. Characteristic periodicities for eight beta -stran ds that comprise the beta -barrel and three alpha -helices were identified. The putative beta -strand I was relatively exposed to solvent, suggesting it does not participate in the formation of the beta -barrel. The beta -str ands A and F contain beta -bulges. The average lambda (max) of emission max ima reveals that strand D is at the edge of the barrel and beta -strand H i nteracts with the main alpha -helical domain. On the basis of the SDTF data , a 3D homology model was constructed for TL and compared to the known crys tallographic structures of RBP and beta -lactoglobulin. The small size and splayed open configuration of the E-F hairpin facilitate access of ligands into the cavity mouth of TL as compared to that of RBP with a long overhang ing loop that restricts access. In the model of TL, four alanine residues a re positioned in the binding site as compared to bulkier residues in the co rresponding positions of beta -lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data sugges t that the smaller side chains of Ala provide more capacity in the cavity o f TL than the bulkier side chains (I56, I71, V92) in the cavity of beta -la ctoglobulin. The structural features provide an explanation for the promisc uous binding characteristics exhibited by TL. SDTF provides a general appro ach for determining the solution structure of many proteins and enhances ho mology modeling in the absence of high sequence identity.