Coupling of dyes to biopolymers by sensitized photooxidation. Affinity labeling of a binding site in bovine serum albumin

Brandt, Johnny; Fredriksson, Maria; Andersson, Lars

doi:10.1021/bi00720a012

Cited by 20 publications

(4 citation statements)

References 22 publications

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“…Neither the addition of UV-irradiated ANS to nonirradiated RecA nor the addition of UV-irradiated RecA to nonirradiated ANS resulted in incorporation of ANS into the protein (data not shown). These results may support a mechanism of photoincorporation similar to photosensitized oxidation, as proposed by Brandt and co-workers (67).…”

Section: Discussionsupporting

confidence: 87%

Probing of DNA-Binding Sites of Escherichia coli RecA Protein Utilizing 1-Anilinonaphthalene-8-sulfonic Acid

Masui

Kuramitsu

1998

Biochemistry

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RecA protein of Escherichia coli plays an essential role in homologous recombination of DNA strands. To analyze the interaction of RecA with single-stranded DNA (ssDNA), we performed a fluorescence competition assay employing 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorescent probe. ANS bound to RecA at three sites, leading to enhancement of ANS fluorescence. Addition of synthetic polynucleotides to the RecA-ANS complex in the absence of a nucleotide quenched the ANS fluorescence, indicating displacement of ANS molecules by ssDNA. Less effective quenching by poly(dA) suggests that the nucleoprotein filament on poly(dA) may differ from those on poly(dT) and poly(dC). A titration experiment with poly(dT) and poly(dA) showed clear stoichiometric binding of 3.5 nucleotides per protein. The site size for poly(dC) was 7.0, which could be explained by the formation of a double helix of poly(dC). ATP and other nucleotides also displaced the ANS. To identify ANS-binding sites, ANS was incorporated into RecA by UV irradiation, and fluorescent peptides were isolated from the proteolytic digest. Sequence analysis suggested that ANS binds to or near the ATP-binding region. These results suggest that the fluorescence quenching and photoincorporation assay using ANS may be useful for the analysis of the interaction of a protein and its ligand.

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Section: Discussionsupporting

confidence: 87%

Probing of DNA-Binding Sites of Escherichia coli RecA Protein Utilizing 1-Anilinonaphthalene-8-sulfonic Acid

Masui

Kuramitsu

1998

Biochemistry

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“…Bis-ANS Can Be Photoincorporated into Proteins and the Adduct Remains Fluorescent. Brandt and co-workers showed that several fluorescent dyes could be coupled to biopolymers through photooxidation (Brandt et al, 1974). In order to see if bis-ANS could be incorporated into proteins, various proteins were irradiated with UV light in the presence of bis-ANS.…”

Section: Resultsmentioning

confidence: 99%

Photoincorporation of 4,4'-Bis(1-anilino-8-naphthalenesulfonic Acid) into the Apical Domain of GroEL: Specific Information from a Nonspecific Probe

Seale¹,

Martinez²,

Horowitz³

1995

Biochemistry

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The use of noncovalent hydrophobic probes such as bis-ANS has become increasingly popular in gaining structural information about protein structure and conformation. While these probes have provided rich information about protein conformation, specific information has been limited. In this report, we extend the usefulness of the probe bis-ANS by showing that it can be covalently photoincorporated into various proteins. Using the chaperonin GroEL, we have shown that it is possible to locate important hydrophobic surfaces through photoincorporation and peptide sequencing. It has been proposed that hydrophobic surfaces on the chaperonin may be responsible for the binding of unfolded polypeptides. We show here that photoincorporation of bis-ANS is able to locate a distinct hydrophobic surface on GroEL. Incorporation of the bis-ANS occurs within a 45 residue fragment of the monomer near the middle of the primary sequence. Interestingly, photoincorporation occurs within this fragment in both tetradecamers and assembly-competent monomers. From the three-dimensional structure of GroEL, this region maps to the apical domain (residues 191-376), which has been implicated in polypeptide binding [Fenton, W. A., Kashi, Y., Furtak, K., & Horwich, A. L. (1994) Nature 371, 614-619]. In addition, the fluorescent properties of the probe are retained including the excitation and emission maxima and the sensitivity to the polarity of its environment. These results suggest that photoincorporated bis-ANS may be a useful probe for protein structure and dynamics.

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“…It serves as the primary carrier of various solutes in plasma, including cations, bilirubin, fatty acids, and therapeutic drugs . There is extensive literature regarding serum albumin’s affinities to various compounds, − denaturation conditions, ,− gelation mechanisms, − and current or potential medical uses. − Albumin hydrogels formed by thermal or chemical cross-linking (e.g., glutaraldehyde) or by polymer-albumin conjugated methods have been reported. ,,,− However, these hydrogel systems typically require either the physical/chemical modification of the albumin or the incorporation of synthetic components into the hydrogel network.…”

Section: Introductionmentioning

confidence: 99%

Electrostatic Unfolding and Interactions of Albumin Driven by pH Changes: A Molecular Dynamics Study

Baler¹,

et al. 2014

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A better understanding of protein aggregation is bound to translate into critical advances in several areas, including the treatment of misfolded protein disorders and the development of self-assembling biomaterials for novel commercial applications. Because of its ubiquity and clinical potential, albumin is one of the best-characterized models in protein aggregation research; but its properties in different conditions are not completely understood. Here, we carried out all-atom molecular dynamics simulations of albumin to understand how electrostatics can affect the conformation of a single albumin molecule just prior to self-assembly. We then analyzed the tertiary structure and solvent accessible surface area of albumin after electrostatically triggered partial denaturation. The data obtained from these single protein simulations allowed us to investigate the effect of electrostatic interactions between two proteins. The results of these simulations suggested that hydrophobic attractions and counterion binding may be strong enough to effectively overcome the electrostatic repulsions between the highly charged monomers. This work contributes to our general understanding of protein aggregation mechanisms, the importance of explicit consideration of free ions in protein solutions, provides critical new insights about the equilibrium conformation of albumin in its partially denatured state at low pH, and may spur significant progress in our efforts to develop biocompatible protein hydrogels driven by electrostatic partial denaturation.

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Coupling of dyes to biopolymers by sensitized photooxidation. Affinity labeling of a binding site in bovine serum albumin

Cited by 20 publications

References 22 publications

Probing of DNA-Binding Sites of Escherichia coli RecA Protein Utilizing 1-Anilinonaphthalene-8-sulfonic Acid

Probing of DNA-Binding Sites of Escherichia coli RecA Protein Utilizing 1-Anilinonaphthalene-8-sulfonic Acid

Photoincorporation of 4,4'-Bis(1-anilino-8-naphthalenesulfonic Acid) into the Apical Domain of GroEL: Specific Information from a Nonspecific Probe

Electrostatic Unfolding and Interactions of Albumin Driven by pH Changes: A Molecular Dynamics Study

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