title: pH-Dependent conformational changes in tear lipocalin by site-directed tryptophan fluorescence.
abstract: Tear lipocalin (TL), a major protein of human tears, binds a broad array of endogenous ligands. pH-dependent ligand binding in TL may have functional implications in tears. Previously, conformational selections of the AB and GH loops have been implicated in ligand binding by site-directed tryptophan fluorescence (SDTF). In this study, SDTF was applied to the AB and GH loops to investigate pH-driven conformational changes relevant to ligand binding. Both loops demonstrate significant but distinct conformational rearrangements over a wide pH range. In the low-pH transition, from 7.3 to 3.0, residues of the GH loop exhibit decreased solvent accessibilities. In acrylamide quenching experiments, the average quenching rate constant (k(q), accessibility parameter) of the residues in the GH loop is decreased approximately 38%, from 2.1 x 10(9) to 1.3 x 10(9) M(-1) s(-1). However, despite the significant changes in accessibilities for some residues in the AB loop, the average accessibility per residue remained unchanged (average k(q) = 1.2 M(-1) s(-1)). Accordingly, the low-pH transition induces conformational changes that reshuffle the accessibility profiles of the residues in the AB loop. A significant difference in the titration curves between the holo and apo forms of the W28 mutant suggests that the protonation states of the residues around position 28 modulate conformational switches of the AB loop relevant to ligand binding.
authors.affiliation: Department of Pathology, UCLA School of Medicine, Jules Stein Eye Institute, 100 Stein Plaza, Los Angeles, California 90095, USA.
authors.forename: Oktay K
authors.initials: OK
authors.lastname: Gasymov
authors.forename: Adil R
authors.initials: AR
authors.lastname: Abduragimov
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Glasgow
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D054835:Lipocalin 1 / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005136:Eye Proteins; D008024:Ligands; D054835:Lipocalin 1; D014364:Tryptophan