The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p -acetyl-L-phenylalanine ( p -AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each containing a single p -AcPhe at a solvent-exposed helix site; the mutants were expressed in amounts qualitatively similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants containing the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in solution. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.

A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUAPyl, a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogues created mutant proteins with distinct spectral properties. This work further expands the structural and chemical diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.

Summary Photocleavage of the polypeptide backbone is potentially a powerful and general method to activate or deactivate functional peptides and proteins with high spatial and temporal resolution. Here we show that 2-nitrophenylalanine is able to photochemically cleave the polypeptide backbone by an unusual cinnoline forming reaction. This unnatural amino acid was genetically encoded in E. coli, and protein containing 2-nitrophenylalanine was expressed and site specifically photocleaved.

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Objective Deficiency in complement factor 5 (C5) protects against arthritis development. However, there is currently no approach translating these findings successfully into arthritis therapy, as by targeting the key component, C5a. Methods We generated an anti-C5a vaccine by incorporating the unnatural amino acid p-nitrophenylalanine (4NPA) at selected sites into murine C5a. C5a-4NPA variants were screened for their immunogenicity on different arthritis susceptiple MHCII backgrounds. A vaccine candidate was tested for its impact on disease in the collagen induced arthritis (CIA) mouse model. Immunity towards endogenous C5a as well as type II collagen was monitored and characterized. Results The replacement of a single tyrosine residue in position 35 (Y35) by 4NPA generated an anti-C5a vaccine, which partly protected mice from developing CIA while strongly ameliorating severity of clinical disease. Although differing in just three atoms from wtC5a, C5aY354NPA induced loss of T and B cell tolerance towards the endogenous protein in mice expressing MHCII H2q molecules. Despite differential B cell epitope recognition, antibodies induced by either wtC5a or C5aY354NPA both neutralized C5a. Thus, anti-wtC5a IgG titers during arthritis priming were potentially critical for disease protection as high titers of C5a-neutralizing antibodies post disease onset were unable to reverse the arthritis course. Conclusion Our results suggest the most effective anti-C5a treatment in arthritis to be accomplished by a preventive vaccination strategy, while conventional biological or small molecule treatments targeting the C5a:C5aR axis risk missing the optimal therapeutic window for intervention during the subclinical priming phase of the disease.