Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity

Gasymov, Oktay K.; Abduragimov, Adil R.; Yusifov, Taleh N.; Glasgow, Ben J.

doi:10.1016/s0167-4838(99)00133-8

Cited by 86 publications

(121 citation statements)

References 32 publications

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“…Recombinant TL is rich with palmitic acid but in contrast to native TL does not contain cholesterol [21,23].…”

Section: Preparation Of Apo-tlmentioning

confidence: 96%

“…Flanking restriction sites for NdeI and BamHI were added to produce the native protein sequence as found in tears but with an initiating methionine [19]. To construct mutant proteins with a single tryptophan, the previously well characterized TL mutant, W17Y, was prepared with oligonucleotides (Universal DNA Inc.) by sequential PCR steps [20,21]. Using this mutant as a template, mutant cDNAs were constructed in which the selected amino acids were additionally substituted with tryptophan or cysteine.…”

Section: Site-directed Mutagenesis and Plasmid Constructionmentioning

confidence: 99%

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Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

Gasymov

Abduragimov

Glasgow

2008

Analytical Biochemistry

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The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the 1 L a and 1 L b components. The relative intensities of the 0-0 transitions of the 1 L b bands correlate linearly with the solvent polarity values . A novel strategy, which utilizes a set of the experimental 1 L b bands, was employed to resolve the near-UV CD spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence as well as structural data of TL. Analysis of the 1 L b bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The "DMSOlike" 1 L b band of Trp CD spectra may be used as a "fingerprint" to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side-chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method we call site-directed circular dichroism is broadly applicable to various proteins to obtain the position-specific data.

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“…Recombinant TL is rich with palmitic acid but in contrast to native TL does not contain cholesterol [21,23].…”

Section: Preparation Of Apo-tlmentioning

confidence: 96%

Section: Site-directed Mutagenesis and Plasmid Constructionmentioning

confidence: 99%

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

Gasymov

Abduragimov

Glasgow

2008

Analytical Biochemistry

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“…Therefore, the mutant proteins have a native fold, and each site-directed mutant reports the features of the native structure. Compared with the wild type of TL or single Cys mutants (K114C and H84C), some single Trp mutants show an expected loss of the positive contribution at 230 nm ( Figure 2) that is attributable to the absence of Trp17 rather than the structural changes (34). The negative deviations of the CD spectra around 230 nm vary among the mutant proteins (e.g., A66W and H84W) relative to that of wild-type TL.…”

Section: Characterization Of Mutant Proteinsmentioning

confidence: 95%

“…DAUDA exhibits little fluorescence in buffer but shows enhancement of fluorescence and a blue shift when bound to tear lipocalin (34). The displacement of DAUDA by ligands is accompanied by a decrease in fluorescence intensity.…”

Section: Competitive Displacement Assays To Assess the Ligand Charge mentioning

confidence: 99%

“…Flanking restriction sites for NdeI and BamHI were added to produce the native protein sequence as found in tears but with an initiating methionine (32). To construct mutant proteins with a single tryptophan, the previously well characterized TL mutant W17Y was prepared with oligonucleotides (Universal DNA Inc., Tigard, OR) by sequential PCR steps (33,34). Using this mutant as a template, mutant cDNAs were constructed in which selected amino acids were additionally substituted with tryptophan or cysteine.…”

Section: Site-directed Mutagenesis and Plasmid Constructionmentioning

confidence: 99%

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Ligand Binding Site of Tear Lipocalin: Contribution of a Trigonal Cluster of Charged Residues Probed by 8-Anilino-1-naphthalenesulfonic Acid

2008

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Human tear lipocalin (TL) exhibits diverse functions, most of which are linked to ligand binding. To map the binding site of TL for some amphiphilic ligands, we capitalized on the hydrophobic and hydrophilic properties of 8-anilino-1-naphthalenesulfonic acid (ANS). In single Trp mutants, resonance energy transfer from Trp to ANS indicates that the naphthalene group of ANS is proximate to Leu105 in the cavity. Binding energies of TL to ANS and its analogues reveal contributions from electrostatic interactions. The sulfonate group of ANS interacts strongly with the nonconserved intracavitary residue Lys114 and less with neighboring residues His84 and Glu34. This trigonal cluster of residues may play a role in the ligand recognition site for some negatively charged ligands. Because many drugs possess sulfonate groups, the trigonal cluster-sulfonate interaction can also be exploited as a lipocalin-based drug delivery mechanism. The binding of lauric acid and its analogues shows that fatty acids assume heterogeneous orientations in the cavity of TL. Predominantly, the hydrocarbon tail is buried in the cavity of TL and the carboxyl group is oriented toward the mouth. However, TL can also interact, albeit relatively weakly, with fatty acids oriented in the opposite direction. As the major lipid binding protein of tears, the ability to accommodate fatty acids in two opposing orientations may have functional implications for TL. At the aqueous-lipid interface, fatty acids whose carboxyl groups are positioned toward the aqueous phase are available for interaction with TL that could augment stability of the tear film.

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Mechanisms and effects of “fat taste” in humans

2014

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Evidence supporting a "taste" cue from fat in the oral cavity continues to accrue. The proposed stimuli for fat taste, non-esterified fatty acids (NEFA), are released from food through hydrolytic rancidity and lipase activity derived from foods or saliva. NEFA must then be released from the food matrix, negotiate the aqueous environment to reach taste cell surfaces, and interact with receptors such as CD36 and GPR120 or diffuse across cell membranes to initiate a taste signal. Knowledge of these processes in non-gustatory tissues should inform understanding of taste responses to NEFA. Additionally, downstream effects of oral triglyceride exposure have been observed in numerous studies. Data specific to effects of NEFA versus triglyceride are scarce, but modified sham feeding trials with triglyceride document cephalic phase responses including elevations in serum lipids and insulin as well as potential, but debated, effects on gut peptides, appetite, and thermogenesis. In this review, we highlight the mechanisms by which NEFA migrate to and interact with taste cells, and then we examine physiological responses to oral fat exposure.

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Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity

Cited by 86 publications

References 32 publications

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

Ligand Binding Site of Tear Lipocalin: Contribution of a Trigonal Cluster of Charged Residues Probed by 8-Anilino-1-naphthalenesulfonic Acid

Mechanisms and effects of “fat taste” in humans

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