Abstract
Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling (SDSL) is an important tool to obtain long-range distance restraints for protein structural research. We here study a variety of azide- and alkyne-bearing noncanonical amino acids (ncAA) in terms of protein single- and double-incorporation efficiency via nonsense suppression, metabolic stability, yields of nitroxide labeling via copper-catalyzed [3 + 2] azide–alkyne cycloadditions (CuAAC), and spectroscopic properties in continuous-wave and double electron–electron resonance measurements. We identify para-ethynyl-l-phenylalanine and para-propargyloxy-l-phenylalanine as suitable ncAA for CuAAC-based SDSL that will complement current SDSL approaches, particularly in cases in which essential cysteines of a target protein prevent the use of sulfhydryl-reactive spin labels.
Highlights
Alternative labeling strategies have been reported that make use of amber stop-codon suppression with noncanonical amino acids (ncAA).[10,11] In this approach, an orthogonal pair of an aminoacyl-tRNA-synthetase (aaRS) and amber suppressor tRNA is co-expressed with the target protein bearing an in-frame amber codon, enabling the co-translational incorporation of the ncAA at user-defined positions directly in cells.[8,12] Here, ncAA can be incorporated that either already contain a paramagnetic center[13,14] or that contain a chemical handle for post-translational bioorthogonal conjugation reactions with a spin label, for example, by azide−alkyne cycloadditions[15−17] or by oxime formation.[18]
We evaluate incorporation efficiencies of the noncanonical amino acids (ncAA) using a generally applicable, polyspecific aaRS/tRNA pair using the Escherichia coli oxidoreductase thioredoxin (TRX) as target protein that contains two essential cysteines as part of its catalytic center
To allow for the flexible incorporation of all ncAA with a single aaRS/tRNA pair, we tested a mutant Methanocaldococcus jannaschii tyrosyl aaRS/tRNA pair previously evolved for the ncAA para-cyanophenylalanine.[32]
Summary
Alternative labeling strategies have been reported that make use of amber stop-codon suppression with noncanonical amino acids (ncAA).[10,11] In this approach, an orthogonal pair of an aminoacyl-tRNA-synthetase (aaRS) and amber suppressor tRNA is co-expressed with the target protein bearing an in-frame amber codon, enabling the co-translational incorporation of the ncAA at user-defined positions directly in cells.[8,12] Here, ncAA can be incorporated that either already contain a paramagnetic center[13,14] or that contain a chemical handle for post-translational bioorthogonal conjugation reactions with a spin label, for example, by azide−alkyne cycloadditions[15−17] or by oxime formation.[18].
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