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

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

doi:10.1016/j.ab.2007.11.002

Cited by 10 publications

(23 citation statements)

References 39 publications

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“…All CD spectra of aqueous solutions of bioconjugates were characterized by a strong negative band around 200 nm and a positive band at 230 nm, with a shoulder at 250 nm, indicating a predominantly unordered conformation. A negative band around 290 nm, most probably caused by interactions between aromatic residues (Trp and daunorubicin), was also present in all spectra [28]. The CD spectra recorded in trifluoroethanol were characterized by an intensive negative band around 200 nm with a shoulder at around 210 nm, a positive band at 230 nm and another one at 250 nm, also indicating a predominantly unordered structure.…”

Section: Secondary Structure Determination By Circular Dichroism Specmentioning

confidence: 77%

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin–GnRH-III bioconjugates

Hegedüs

Manea

Orbán

et al. 2012

European Journal of Medicinal Chemistry

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Here we report on the synthesis and biochemical characterization (enzymatic stability, cellular uptake, in vitro antitumor activity, membrane interaction and GnRH-receptor binding affinity) of novel short-chain fatty acid (SCFA) acylated daunorubicin-GnRH-III bioconjugates, which may serve as drug delivery systems for targeted cancer chemotherapy. Ser in position 4 of GnRH-III was replaced by Lys, followed by the acylation of its ε-amino group with various fatty acids. SCFAs are potentially chemoprotective agents by suppressing the growth of cancer cells and therefore may enhance the antitumor activity of the bioconjugates. We found that all synthesized bioconjugates had high cytostatic effect in vitro, were stable in cell culture medium for 6 h and degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked daunorubicin-Lys as the smallest active metabolite. In the presence of α-chymotrypsin, all compounds were digested, the degradation rate strongly depending on the type of fatty acid. The bioconjugate containing Lys(nBu) in position 4 was taken up most efficiently by the cancer cells and exerted higher in vitro cytostatic effect than the previously developed GnRH-III( 4 Lys(Ac), 8 Lys(Dau=Aoa)) or the parent GnRH-III(Dau=Aoa) bioconjugate. Our results could be explained by the increased binding affinity of the newly developed compound containing Lys(nBu) to the GnRH receptors.

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Section: Secondary Structure Determination By Circular Dichroism Specmentioning

confidence: 77%

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin–GnRH-III bioconjugates

Hegedüs

Manea

Orbán

et al. 2012

European Journal of Medicinal Chemistry

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“…The wavelength positions of the vibrational bands of the CD spectra of Trp and Tyr depend on their nearest environment (polarity, nearby charged group, etc.) [49, 50]. Therefore, the features of the near-UV CD spectra of proteins reflect specific tertiary structure and are unique for each protein.…”

Section: Resultsmentioning

confidence: 99%

The conserved disulfide bond of human tear lipocalin modulates conformation and lipid binding in a ligand selective manner

Gasymov

Abduragimov

Glasgow

2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The primary aim of this study is the elucidation of the mechanism of disulfide induced alteration of ligand binding in human tear lipocalin (TL). Disulfide bonds may act as dynamic scaffolds to regulate conformational changes that alter protein function including receptor-ligand interactions. A single disulfide bond, (Cys61-Cys153), exists in TL that is highly conserved in the lipocalin superfamily. Circular dichroism and fluorescence spectroscopies were applied to investigate the mechanism by which disulfide bond removal effects protein stability, dynamics and ligand binding properties. Although the secondary structure is not altered by disulfide elimination, TL shows decreased stability against urea denaturation. Free energy change (ΔG0) decreases from 4.9± 0.2 to 2.1± 0.3 kcal/mol with removal of the disulfide bond. Furthermore, ligand binding properties of TL without the disulfide vary according to the type of ligand. The binding of a bulky ligand, NBD-cholesterol, has a decreased time constant (from 11.8± 0.2 to 3.3 s). In contrast, the NBD-labeled phospholipid shows a moderate decrease in the time constant for binding, from 33.2± 0.2 to 22.2± 0.4 s. FRET experiments indicate that the hairpin CD is directly involved in modulation of both ligand binding and flexibility of TL. In TL complexed with palmitc acid (PA-TL), the distance between the residues 62 of strand D and 81 of loop EF is decreased by disulfide bond reduction. Consequently, removal of the disulfide bond boosts flexibility of the protein to reach a CD-EF loop distance (24.3 Å, between residues 62 and 81), which is not accessible for the protein with an intact disulfide bond (26.2 Å). The results suggest that enhanced flexibility of the protein promotes a faster accommodation of the ligand inside the cavity and energetically favorable ligand-protein complex.

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“…7 In addition, fluorescence lifetime analysis of Trp130 indicates that its side chain assumes predominantly (84%) one rotamer ( t ). Trp residues in the α-helix conformation may assume only two ( g – (+60°) and t (180°)) out of three possible χ 1 rotamers.…”

Section: Discussionmentioning

confidence: 99%

Probing Tertiary Structure of Proteins Using Single Trp Mutations with Circular Dichroism at Low Temperature

2014

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Trp is the most spectroscopically informative aromatic amino acid of proteins. However, the near-UV CD spectrum of Trp is complicated because the intensity and sign of 1La and 1Lb bands vary independently. To resolve vibronic structure and gain site-specific information from complex spectra, deconvolution was combined with cooling and site-directed tryptophan substitution. Low temperature near-UV CD was used to probe the local tertiary structure of a loop and α-helix in tear lipocalin. Upon cooling, the enhancement of the intensities of the near-UV CD was not uniform, but depends on the position of Trp in the protein structure. The most enhanced 1Lb band was observed for Trp at position 124 in the α-helix segment matching the known increased conformational mobility during ligand binding. Some aspects of the CD spectra of W28 and W130 were successfully linked to specific rotamers of Trp previously obtained from fluorescence lifetime measurements. The discussion was based on a framework that the magnitude of the energy differences in local conformations governs the changes in the CD intensities at low temperature. The Trp CD spectral classification of Strickland was modified to facilitate the recognition of pseudo-peaks. Near-UV CD spectra harbor abundant information about the conformation of proteins that site directed Trp CD can report.

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

Cited by 10 publications

References 39 publications

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin–GnRH-III bioconjugates

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin–GnRH-III bioconjugates

The conserved disulfide bond of human tear lipocalin modulates conformation and lipid binding in a ligand selective manner

Probing Tertiary Structure of Proteins Using Single Trp Mutations with Circular Dichroism at Low Temperature

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