RET and anisotropy measurements establish the proximity of the conserved Trp17 to Ile98 and Phe99 of tear lipocalin.
Previous studies suggest that the conserved Trp17 on strand A of TL has a role in lipocalin stability and interacts, directly or indirectly, with Ile98 and Phe99 on strand G to influence ligand binding. Here, we determined the proximity of Trp17 to Ile98 and Phe99. Time-resolved fluorescence experiments showed resonance energy transfer between tryptophans at positions 17 and 98. In addition, an exciton effect was discovered in CD experiments resulting from interactions of the excited states of these tryptophans. Fluorescence anisotropy values of mutants containing two tryptophans (positions 99/17 and 98/17) were lower than expected in the absence of RET, confirming that these residues are proximate in tear lipocalin. The data support a model of tear lipocalin in which Trp17 and Phe99 are close together deep in the cavity and participate in an internal hydrophobic cluster. Ile98 is proximate to Trp17 but faces toward the outside of the cavity and in the model is part of an external hydrophobic patch. Comparison with beta-lactoglobulin suggests that these motifs may have an important influence on protein stability and ligand binding in other members of the lipocalin family.
Gasymov
Oktay K
OK
Department of Pathology, School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA.
Abduragimov
Adil R
AR
Yusifov
Taleh N
TN
Glasgow
Ben J
BJ
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D006801:Humans; D007532:Isoleucine; D054835:Lipocalin 1; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan
D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; C518443:LCN1 protein, human; D054835:Lipocalin 1; D011994:Recombinant Proteins; D007532:Isoleucine; D010649:Phenylalanine; D014364:Tryptophan