The Mechanism of Photoaffinity Labeling

Ruoho, Arnold E.; Kiefer, H.; Roeder, Phoebe E.; Singer, S. J.

doi:10.1073/pnas.70.9.2567

Cited by 139 publications

(71 citation statements)

References 14 publications

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“…The observed lifetimes, 10-7 to 10-9 sec, are very short compared with the 10-3-sec lifetimes of typical aromatic nitrenes (26). : The short lifetime will ensure that the photoac--tivated species does not wander away from its proper target before reacting (27).…”

Section: Och3mentioning

confidence: 99%

High yield photoreagents for protein crosslinking and affinity labeling

Jelenc

Cantor

Simon

1978

Proc. Natl. Acad. Sci. U.S.A.

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4-Nitrophenyl ethers are proposed as new hig-yield photoreagents for protein crosslinking and affinity labeling. These are totally unreactive in the dark under biological conditions, but react quantitatively with &mines at pH 8 upon irradiation with 368nm light. The reaction of monoalkoxy-p-nitrobenzenes with an amine yields the corresponding free aicobol and substituted nitrophenylamine. In essence, the nitrophenyl group is transferred from the alcohol to the amine. Bifunctional affinity reagents of this type could be especially useful for placing the pnitrophenyl chromophore adjacent to a binding site without blocking it. The corresponding 2-methoxy4nitrophenyl ethers react with amines by displacement of the methoxyl group. Thus, bifunctional reagents of this class could be photocrosslinkers. A maleimide-containing 2-methoxy-4-nitrophenyl ether was attached to human fetal hemoglobin at 'y-cysteine F9 stoichiometrically. Subseuent ultraviolet irradiation yielded a y-y crosslinked hemoglobin in 80% yield. The oxygenation properties of the derivative indicate that it is locked in a high afrinity conformation and that all cooperativity is lost.Crosslinking and affinity labeling of biological macromolecules have been used with increasing frequency for structural and functional studies. The first reagents employed in these techniques were derived from common acylating and alkylating reagents (1, 2). The fate of such reagents in solutions of bio-

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Section: Och3mentioning

confidence: 99%

High yield photoreagents for protein crosslinking and affinity labeling

Jelenc

Cantor

Simon

1978

Proc. Natl. Acad. Sci. U.S.A.

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“…These studies have used the small ribosomal subunit of different bacterial species, and there seems to be no agreement on the binding site of tetracycline on the 16S rRNA (12). In addition, many of these studies were conducted using methods that are prone to introduce errors, such as crossreactions with products of photolysis during photoaffinity labeling (9,59). Hence, the proposed binding sites may not represent the actual target sites in vivo.…”

Section: Mechanism(s) Of Action Of the Tetracyclines: The Journey So Farmentioning

confidence: 99%

rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines

Chukwudi

2016

Antimicrob Agents Chemother

151

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The tetracycline antibiotics are known to be effective in the treatment of both infectious and noninfectious disease conditions. The 16S rRNA binding mechanism currently held for the antibacterial action of the tetracyclines does not explain their activity against viruses, protozoa that lack mitochondria, and noninfectious conditions. Also, the mechanism by which the tetracyclines selectively inhibit microbial protein synthesis against host eukaryotic protein synthesis despite conservation of ribosome structure and functions is still questionable. Many studies have investigated the binding of the tetracyclines to the 16S rRNA using the small ribosomal subunit of different bacterial species, but there seems to be no agreement between various reports on the exact binding site on the 16S rRNA. The wide range of activity of the tetracyclines against a broad spectrum of bacterial pathogens, viruses, protozoa, and helminths, as well as noninfectious conditions, indicates a more generalized effect on RNA. In the light of recent evidence that the tetracyclines bind to various synthetic double-stranded RNAs (dsRNAs) of random base sequences, suggesting that the double-stranded structures may play a more important role in the binding of the tetracyclines to RNA than the specific base pairs, as earlier speculated, it is imperative to consider possible alternative binding modes or sites that could help explain the mechanisms of action of the tetracyclines against various pathogens and disease conditions.

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“…Light induces the formation of reactive intermediates that attack nearby groups to form covalent bonds. The effectiveness of a photoaffinity agent, therefore, depends upon its affinity for the binding site in the dark and upon the half-lives of these photogenerated intermediates (4,24). The half-life of the ligandbinding site complex must be considerably longer than the halflives of the intermediates to achieve specific labeling.…”

mentioning

confidence: 99%

Azido Auxins

Jones¹,

Melhado²,

Ho³

et al. 1984

Plant Physiol.

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The binding constants of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic acid (4-, 5-, and 6-N3IAA), and of the photoproducts of 5-N3IAAto the naphthalene-l-acetic acid (NAA) binding sites of Zea mays L. WF9 x BR38 were determined to evaluate the potential of these analogs as photoaffinity labeling agents. We have found that 4-and 5-N3IAA bind to these sites with affinities similar to that of IAA, while 6-N3IAA and the photoproducts of 5-N3IAA bind less tightly. This 3School of Chemical Sciences. 4Abbreviations: NAA, naphthalene-l-acetic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; NAA, naphthalene-1-acetic acid; N3IAA, azidoindole-3-acetic acid; 4-N3IAA, 4-azidoindole-3-acetic acid; 5-N3IAA, 5-azidoindole-3-acetic acid; 6-N3IAA, 6-azidoindole-3-acetic acid; RAC, ratio of association constants.that avoids the requirement that conditions for equilibrium binding be maintained while searching among the membrane proteins for specific auxin-binding sites. Once binding proteins are labeled, they can be isolated using standard techniques without the need to maintain equilibrium binding conditions. Therefore, the advantage of photoaffinity labeling is that binding proteins can still be isolated after they have lost the capacity for binding. Photoaffinity analogs bind reversibly to a site in the dark but can be caused to bind irreversibly by irradiation with light. Light induces the formation of reactive intermediates that attack nearby groups to form covalent bonds. The effectiveness of a photoaffinity agent, therefore, depends upon its affinity for the binding site in the dark and upon the half-lives of these photogenerated intermediates (4,24). The half-life of the ligandbinding site complex must be considerably longer than the halflives of the intermediates to achieve specific labeling.The only previous attempt to label the auxin site by affinity labeling was by Venis (26). He tested the usefulness of the diazonium salts of two auxin analogs, 2-chloro-4-aminophenoxyacetic acid and 2,5-dichloro-3-aminobenzoic acid, and reached tentative conclusions about the involvement of several amino acids in the auxin active site. The potential photoaffinity labeling agents 4-azido-2-chlorophenoxyacetic acid and 3-azido-5-chlorophenoxyacetic acid were found to have only moderate auxin activity (15), unlike the azidoindole-3-acetic acids here described. Spring and Hager (25) have recently demonstrated that sesquiterpene lactones specifically label important growth sites only when an auxin is also bound. The experiments provide an example of a nonequilibrium binding technique useful in auxin receptor research and are important because the results support a molecular model for auxin binding. The authors propose that auxin induces the receptor to undergo a conformational change, important for cell growth. Most significantly, the technique offers an independent assay for identifying auxin receptors and could be useful in conjunction with an independent approach such as we propose in this report.We ...

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The Mechanism of Photoaffinity Labeling

Cited by 139 publications

References 14 publications

High yield photoreagents for protein crosslinking and affinity labeling

High yield photoreagents for protein crosslinking and affinity labeling

rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines

Azido Auxins

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