Intracavitary ligand distribution in tear lipocalin by site-directed tryptophan fluorescence.
Site-directed tryptophan fluorescence has been successfully used to determine the solution structure of tear lipocalin. Here, the technique is extended to measure the binding energy landscape. Single Trp mutants of tear lipocalin are bound to the native ligand and an analogue tagged with a quencher group to both populate and discriminate the excited protein states. Steady-state and time-resolved fluorescence quenching data reveal the intracavitary state of the ligand. The static components of fluorescence quenching identify the residues where nonfluorescence complexes form. An asymmetric distribution of the ligand within the cavity reflects the complex energy landscape of the excited protein states. These findings suggest that the excited protein states are not unique but consist of many substates. The roughness of the binding energy landscape is about 2.5kBT. The excited protein states originate primarily from conformational selections of loops AB and GH, a portal region. In contrast to static quenching, the dynamic components of fluorescence quenching by the ligand are relevant to both local side chain and ligand dynamics. Apparent bimolecular rate constants for collisional quenching of Trp by the nitroxide moiety are approximately 1 / 5 x 10(12) M(-1) s(-1). Estimations made for effective ligand concentrations establish actual rate constants on the order of 12 x 10(9) M(-1) s(-1). Prior to exit from the cavity of the protein, ligands explore binding sites in nanoseconds. Although microsecond fluctuations are rate-limiting processes in ligand binding for many proteins, accompanying nanosecond motion may be necessary for propagation of ligand binding.
Gasymov
Oktay K
OK
Department of Pathology, UCLA School of Medicine, Jules Stein Eye Institute, 100 Stein Plaza, Los Angeles, California 90095, USA.
Abduragimov
Adil R
AR
Glasgow
Ben J
BJ
D001665:Binding Sites; D003619:Dansyl Compounds / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008024:Ligands; D054835:Lipocalin 1 / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry*
D003619:Dansyl Compounds; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008024:Ligands; D054835:Lipocalin 1; D014364:Tryptophan