3‐Nitrotyrosine as a spectroscopic probe for investigating protein–protein interactions

Filippis, Vincenzo De; Frasson, Roberta; Fontana, Angelo

doi:10.1110/ps.051957006

Cited by 63 publications

(90 citation statements)

References 59 publications

(132 reference statements)

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“…The major physicochemical difference between tyrosine and nitrotyrosine is the decrease in pK a of the phenolic AOH group from 10.1 in tyrosine to 6.8 in nitrotyrosine. 39 This may result in different ionization efficiencies between nitrated and unmodified peptides. However, like phenolic AOH group of tyrosine, the majority of phenolic AOH group of nitrotyrosine should be protonated under the acidic conditions used (pH < 2.5) for LC-MS/MS analysis; therefore, the lower pK a value of nitrotyrosine is unlikely to affect significantly to the ionization efficiencies of nitrated peptides.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

Palamalai¹,

Miyagi²

2010

Protein Science

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifaceted protein that is involved in numerous processes including glycolysis, translational silencing, transcriptional regulation of specific genes, and acting as a nitric oxide sensor. The precise mechanism on how GAPDH is targeted to these different roles is unclear but believed to involve specific posttranslational modification to the protein. Numerous studies have demonstrated that GAPDH is a target for tyrosine nitration. However, the site of modification and the molecular consequence have not been defined. Rabbit GAPDH with a reversibly protected catalytic cysteine was nitrated in vitro with tetranitromethane, resulting in complete loss of GAPDH catalytic activity. Nitration was estimated as 0.32 mol of nitrotyrosine residue per mole of GAPDH. Mass spectrometry analysis of nitrated GAPDH indicated that Tyr311 and Tyr317 were the sole sites of nitration. The X-ray crystal structure revealed that the distances between Tyr311 and Tyr317 and the cofactor nicotinamide adenine dinucleotide (NAD 1 ) were less than 7.2 and 3.7 Å , respectively, implying that nitration of these two residues may affect NAD 1 binding. This possibility was assessed using an NAD 1 binding assay, which showed that nitrated GAPDH was incapable of binding NAD 1 . Thus, these results strongly suggest that Tyr311 and Tyr317 nitration prohibits NAD 1 binding, leading to the loss of catalytic activity.

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Section: Discussionmentioning

confidence: 99%

Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

Palamalai¹,

Miyagi²

2010

Protein Science

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“…The proton may be transferred from a lysine side-chain, however it is worth noting that addition of a nitro-group to tyrosine increases the acidity of the phenolic hydrogen (pK a 7.2 [45]. At acidic pH the hydroxyl group remains uncharged but is involved in hydrogen bonding to the neighboring nitration modification [46]. Hence possibly proton transfer from the phenol group occurs.…”

Section: Loss Of Neutrals Observed Following Ecd Of Nitrated Peptidesmentioning

confidence: 99%

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

Jones

Mikhailov

Iniesta

et al. 2010

J. Am. Soc. Mass Spectrom.

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In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains. (J Am Soc Mass Spectrom 2010, 21, 268 -277)

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“…The spectrum of free P2NT1 presents a marginally weak negative signal in the 300-390-nm range and a low-intensity positive band at 280 nm, assigned to the 1 A 1g ! 1 B 1u transition of NT (Meloun et al 1968;De Filippis et al 2006), which acquires some rotational strength because of the presence of several rigid prolines that stabilize the PP II conformation.…”

Section: Circular Dichroismmentioning

confidence: 99%

“…In particular, iodine-containing molecules are known to act as collisional quenchers of Trp fluorescence by promoting nonradiative decay of the excited singlet state through intersystem crossing to an excited triplet state (Berlman 1973;Lakowicz 1999). The nonfluorescent NT, on the other hand, which absorbs radiation in a pH-dependent manner in the wavelength range where Tyr and Trp emit fluorescence, has proven to be an efficient acceptor in energy transfer processes, such as those occurring in protein folding (Rischel and Poulsen 1995;Tcherkasskaya and Ptitsyn 1999) and molecular recognition (De Filippis et al 2006).…”

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o‐Nitrotyrosine andp‐iodophenylalanine as spectroscopic probes for structural characterization of SH3 complexes

et al. 2007

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High-throughput screening of protein-protein and protein-peptide interactions is of high interest both for biotechnological and pharmacological applications. Here, we propose the use of the noncoded amino acids o-nitrotyrosine and p-iodophenylalanine as spectroscopic probes in combination with circular dichroism and fluorescence quenching techniques (i.e., collisional quenching and resonance energy transfer) as a means to determine the peptide orientation in complexes with SH3 domains. Proline-rich peptides bind SH3 modules in two alternative orientations, according to their sequence motifs, classified as class I and class II. The method was tested on an SH3 domain from a yeast myosin that is known to recognize specifically class I peptides. We exploited the fluorescence quenching effects induced by o-nitrotyrosine and p-iodophenylalanine on the fluorescence signal of a highly conserved Trp residue, which is the signature of SH3 domains and sits directly in the binding pocket. In particular, we studied how the introduction of the two probes at different positions of the peptide sequence (i.e., N-terminally or C-terminally) influences the spectroscopic properties of the complex. This approach provides clearcut evidence of the orientation of the binding peptide in the SH3 pocket. The chemical strategy outlined here can be easily extended to other protein modules, known to bind linear sequence motifs in a highly directional manner.Keywords: SH3 domains; proline-rich peptides; o-nitrotyrosine; p-iodophenylalanine; protein recognition; noncoded amino acids; fluorescence energy transfer; quenching; circular dichroism Supplemental material: see www.proteinscience.orgThe central role played by molecular interactions in the functioning of all biological systems requires a detailed structural understanding of how recognition takes place (Jones and Thornton 1996;Pawson and Nash 2003). Complete structural determination of protein complexes can, however, be time-demanding, since with the current technical tools it is often necessary to undertake de novo structure determinations even when the structures of all the interacting partners are known individually. It is therefore increasingly important to develop new high-throughput approaches that may allow us to grasp quickly at least the basic features of a complex without its detailed and lengthy structural description. Abbreviations: Standard single-or three-letter abbreviations were used for natural amino acids; CD, circular dichroism; DIEA, diisopropylethylamine; EDT, ethanedithiol; ESI, electrospray ionization; Fmoc, 9-fluorenyl-methyloxycarbonyl; FRET, fluorescence resonance energy transfer; IF, p-iodophenylalanine; IFE, inner filter effect; HATU, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; Myo3, isoform 3 of myosin from the yeast Saccharomyces cerevisiae; Myo3-SH3, SH3 domain of Myo3;

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3‐Nitrotyrosine as a spectroscopic probe for investigating protein–protein interactions

Cited by 63 publications

References 59 publications

Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

o‐Nitrotyrosine andp‐iodophenylalanine as spectroscopic probes for structural characterization of SH3 complexes

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