Abstract8-anilino-1-naphthalenesulfonic acid (ANS) is believed to strongly bind cationic groups of proteins and polyamino acids through ion pair formation. A paucity of data exists on the fluorescent properties of ANS in these interactions. ANS binding to arginine and lysine derivatives was studied by fluorescence and circular dichroism spectroscopies to augment published information attained by isothermal titration calorimetry (ITC).Fluorescence enhancement with a hypsochromic shift results from the interaction of the charged group of lysine and arginine with the sulfonate group of ANS. Ion pairing between Arg (or Lys) and the sulfonate group of ANS reduce the intermolecular charge transfer (CT) rate constant that leads to enhancement of fluorescence. A positive charge near the -NH group of ANS changes the intramolecular CT process producing a blue shift of fluorescence. The Arg side chain compared to that of Lys more effectively interacts with both the -NH and sulfonate groups of ANS. ANS binding also induces a random coil-alpha helix transition in poly-Arg.Our data, in contrast to ITC results, indicate that electrostatic interactions between ANS derivatives and positively charged side chains do not account for binding affinity in the micromolar range. In addition to ion pairing complementary interactions, such as van der Waals, should be considered for high affinity (K d < 1mM) external binding sites of proteins.

The solution structure of human TL was deduced from the position of the emission peaks after site-directed tryptophan fluorescence (SDTF). The fluorescent amino acid tryptophan was sequentially substituted for each native amino acid in the sequence. Characteristic periodicities for eight beta-strands 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-strands A and F contain beta-bulges. The average lambda(max) of emission maxima reveals that strand D is at the edge of the barrel and beta-strand H interacts 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 crystallographic 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 overhanging loop that restricts access. In the model of TL, four alanine residues are positioned in the binding site as compared to bulkier residues in the corresponding positions of beta-lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data suggest that the smaller side chains of Ala provide more capacity in the cavity of TL than the bulkier side chains (I56, I71, V92) in the cavity of beta-lactoglobulin. The structural features provide an explanation for the promiscuous binding characteristics exhibited by TL. SDTF provides a general approach for determining the solution structure of many proteins and enhances homology modeling in the absence of high sequence identity.

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Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.