ANS fluorescence: Potential to augment the identification of the external binding sites of proteins

Gasymov, Oktay K.; Glasgow, Ben J.

doi:10.1016/j.bbapap.2007.01.002

Cited by 348 publications

(247 citation statements)

References 18 publications

Supporting

Mentioning

241

Contrasting

Unclassified

Order By: Relevance

“…Fluorescence enhancement upon binding to proteins can result from ion pairing interactions, hydrophobic interactions, restricted mobility or any combination of these factors [33][34][35][36]. EPR spectra of spin labeled G-strand mutants complexed with ANS in molten globule state are shown in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

Molten globule state of tear lipocalin: ANS binding restores tertiary interactions

Gasymov

Abduragimov

Glasgow

2007

Biochemical and Biophysical Research Communications

Self Cite

View full text Add to dashboard Cite

Tear lipocalin (TL) may stabilize the lipid layer of tears through a molten globule state triggered by low pH. EPR spectroscopy with site directed spin labeling, revealed the side chain mobility of residues on the G-strand of TL in a molten globule state; the G-strand retains β-sheet structure. All of the side chains of G strand residues become more loosely packed, especially residues 96-99. In contrast, the highly mobile side chain of residue 95 on the F-G loop, becomes tightly packed. ANS binding to TL in a molten globule state reestablishes tight packing around side chains that are oriented both inside and outside of the barrel. Unlike RBP and BLG; TL has no disulfide bond between G and H strands. It is likely that the central β-sheet in the molten globule state of lipocalins is stabilized by its interactions with the main α-helix, rather than the interstrand disulfide bond.

show abstract

Section: Resultsmentioning

confidence: 99%

Molten globule state of tear lipocalin: ANS binding restores tertiary interactions

Gasymov

Abduragimov

Glasgow

2007

Biochemical and Biophysical Research Communications

Self Cite

View full text Add to dashboard Cite

show abstract

“…Both the binding and fluorescence properties of ANS are influenced strongly by local environmental variables, which include not only hydrophobicity but also neighboring charged groups and the solvent polarity and viscosity (43,44,83). Interaction of positively charged arginine or lysine side chains with the sulfonate group of ANS (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…ANS is a small amphipathic dye (Fig. 1B) that has been used as a sensitive probe to detect hydrophobic pockets on protein surfaces (41)(42)(43)(44). Free ANS exhibits only weak fluorescence that is maximal near 520 nm, but when ANS binds to a hydrophobic site in a partially or fully folded protein, the fluorescence peak increases in amplitude and shifts to a shorter wavelength (42).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

Tiwari

Liba

Sohn

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu 2؉ , even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.The sequence of events by which more than 100 mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1) 3 cause familial forms of amyotrophic lateral sclerosis (ALS) is unknown. Studies of purified SOD1 proteins and cellular or rodent models of SOD1-linked ALS suggest that impaired metal ion binding or misfolding of mutant SOD1 proteins in the cellular environment may be related to their toxicity (1-10). Available evidence suggests that partially unfolded mutant SOD1 species could contribute to motor neuron death by promoting abnormal interactions that produce cellular dysfunction (11-16).In previous studies we characterized physicochemical properties of 14 different biologically metallated ALS SOD1 mutants (17) and demonstrated altered thermal stabilities of these mutants compared with wild-type (WT) SOD1 (18). These "asisolated" SOD1 proteins, which contain variable amounts of copper and zinc, were broadly grouped into two classes based on their ability to incorporate and retain metal ions with high affinity. WT-like SOD1 mutants retain the ability to bind copper and zinc ions and exhibit dismutase activity similar to the normal enzyme, whereas metal binding region (MBR) mutants are significantly deficient in copper and/or zinc (17, 19). We also observed that ALS-associated SOD1 mutants were more susceptible than the WT enzyme to reduction of the intrasubunit disulfide bond between . The significance of these results is that even WT-like mutants, whic...

show abstract

“…Previously, it has been shown that the electrostatic interaction alone is not sufficient to provide high binding affinity for ANS (K d < 1 mM) (44). For internal ANS binding, in addition to the electrostatic interaction, hydrophobic and van der Waals interaction should be considered.…”

Section: Binding Experiments With Ans 1npn and Tns: Electrostatic Cmentioning

confidence: 99%

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

2008

Self Cite

View full text Add to dashboard Cite

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.

show abstract

ANS fluorescence: Potential to augment the identification of the external binding sites of proteins

Cited by 348 publications

References 18 publications

Molten globule state of tear lipocalin: ANS binding restores tertiary interactions

Molten globule state of tear lipocalin: ANS binding restores tertiary interactions

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

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

Contact Info

Product

Resources

About