Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli . Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t 1/2 = 0.4 h) reduction of 3-NT to 3-aminotyrosine (3-AT) was observed. When screening for potential mechanisms, we found that 3-NT can be reduced enzymatically to 3-AT, whilst biologically relevant low molecular weight reductants, such as NADPH or GSH, did not affect 3-NT. A genetic screen for E.coli proteins, involved in the observed 3-NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3-NT reduction as a broadly-relevant pathway in E.coli . In conclusion, incorporation of 3-NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites. The potential reduction of the 3-NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining pure nitrated protein.
Adenosine triphosphate (ATP) probes modified with fluorescence dyes that change their fluorescence properties upon cleavage are an interesting tool for monitoring enzymatic ATP turnover. As a readout parameter, fluorescence lifetime is attractive because it is nearly independent of concentration. In our study, we synthesised and investigated fifteen different ATP analogues, in which the fluorophores were attached to the γ-phosphate of ATP. All analogues showed distinctly different fluorescence lifetimes compared to the corresponding values of the free fluorophores. Both increases and decreases in fluorescence lifetime were observed upon attachment to ATP. To shed light on the photophysical processes governing the lifetime changes, we performed photoelectron spectroscopy in air (PESA) to determine HOMO energy levels and time-resolved fluorescence spectroscopy to obtain rate constants. We present evidence that fluorescence quenching in the compounds tested is dynamic and attributed to photoinduced electron transfer (PET), whereas fluorescence lifetime increases are caused by stacking interactions between chromophore and the nucleobase reducing non-radiative relaxation. Finally, we demonstrate that enzymatic cleavage of the ATP analogues presented can be followed by continuous monitoring of fluorescence lifetime changes.
Pulsed electron–electron double resonance spectroscopy (known as PELDOR or DEER) has recently become a very popular tool in structural biology. The technique can be used to accurately measure distance distributions within macromolecules or macromolecular complexes, and has become a standard method to validate structural models and to study the conformational flexibility of macromolecules. It can be applied in solution, in lipid environments or even in cells. Because most biological macromolecules are diamagnetic, they are normally invisible for PELDOR spectroscopy. To render a particular target molecule accessible for PELDOR, it can be engineered to contain only one or two surface‐exposed cysteine residues, which can be efficiently spin‐labelled using thiol‐reactive nitroxide compounds. This method has been coined “site‐directed spin labelling” (SDSL) and is normally straight‐forward. But, SDSL can be very challenging for proteins with many native cysteines, or even a single functionally or structurally important cysteine residue. For such cases, alternative spin labelling techniques are needed. Here we describe the concept of “inhibitor‐directed spin labelling” (IDSL) as an approach to spin label suitable cysteine‐rich proteins in a site‐directed and highly specific manner by employing bespoke spin‐labelled inhibitors. Advantages and disadvantages of IDSL are discussed.
Simple andr obust assays to monitore nzymatic ATPc leavage with high efficiency in real-time are scarce.T o address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra-and pentaphosphate analogues of ATP. The novel ATPa naloguesb ear -i nc ontrast to earlier reports -o nly as ingle acridone-based dye at the terminal phosphate group. The dye's fluorescencei sq uenched by the adeninec omponent of the ATPa nalogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety.T hereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiq-uitin-activating enzymesU BA1 and UBA6 which represents the first step in an enzymatic cascadel eading to the covalent attachment of ubiquitin to substrate proteins,aprocess that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor forU BA1/UBA6 very much dependso nt he length of the phosphate chain of the ATPa nalogue:t riphosphatesa re used poorly while pentaphosphates are most efficiently processed. Notably,t he novel pentaphosphate-harbouring ATPa nalogues upersedes the efficiency of recently reported dual-dye labelleda nalogues and thus, is ap romising candidate for broad applications.
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