3-Nitrotyrosine as a spectroscopic probe for investigating protein protein interactions.

Protein Science : a Publication of the Protein Society
Vincenzo De FilippisAngelo Fontana

Abstract

3-Nitrotyrosine (NT) is approximately 10(3)-fold more acidic than Tyr, and its absorption properties are strongly pH-dependent. NT absorbs radiation in the wavelength range where Tyr and Trp emit fluorescence (300-450 nm), and it is essentially nonfluorescent. Therefore, NT may function as an energy acceptor in resonance energy transfer (FRET) studies for investigating ligand protein interactions. Here, the potentialities of NT were tested on the hirudin thrombin system, a well-characterized protease inhibitor pair of key pharmacological importance. We synthesized two analogs of the N-terminal domain (residues 1-47) of hirudin: Y3NT, in which Tyr3 was replaced by NT, and S2R/Y3NT, containing the substitutions Ser2 --> Arg and Tyr3 --> NT. The binding of these analogs to thrombin was investigated at pH 8 by FRET and UV/Vis-absorption spectroscopy. Upon hirudin binding, the fluorescence of thrombin was reduced by approximately 50%, due to the energy transfer occurring between the Trp residues of the enzyme (i.e., the donors) and the single NT of the inhibitor (i.e., the acceptor). The changes in the absorption spectra of the enzyme inhibitor complex indicate that the phenate moiety of NT in the free state becomes protonated to ph...Continue Reading

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