No abstract

Control of the spatial arrangement of proteins on surfaces is essential in a number of emerging technologies, including protein microarrays, biosensors, 1 tissue engineering, and regenerative medicine. 2 Patterning is also a powerful tool in cell biology, wherein cell arrays are used to elucidate the factors that mediate migration, proliferation, and cell-cell interactions. 3 Although photolithography holds a preeminent place as a patterning method in the microelectronics industry, optical lithography of proteins has been hampered by the need either to use traditional chemical photoresists or to modify proteins chemically by attachment of photoreactive functional groups; both methods can compromise protein function. 4Production of a protein "photoresist" without the need for post-translational chemical modification would require an intrinsically photoreactive protein. Recently, the incorporation of photo-reactive, non-canonical amino acids into proteins via site-specific 5 and residuespecific techniques has been reported. 6 Here we describe the microbial expression of artificial proteins bearing the photosensitive non-canonical amino acid para-azidophenylalanine (pN 3 Phe). The recombinant proteins, designated artificial extracellular matrix proteins with aryl azides (aECM-N 3 ), belong to a family of engineered proteins designed to exhibit mechanical properties similar to those of native elastins 7 and to support adhesion of mammalian cells through cell-binding domains (CS5 or RGD) derived from fibronectin ( Fig 1A). 8 These proteins can be crosslinked efficiently upon irradiation at 365 nm. The physical properties of the crosslinked films can be controlled by changing the pN 3 Phe content, and thin films can be patterned on surfaces via photolithographic techniques. We demonstrate the utility of the method by creating cell arrays through selective cell attachment to photolithographically prepared protein patterns. aECM-N 3 variants were expressed in Escherichia coli cultures supplemented with pN 3 Phe (Supporting Information). Incorporation of pN 3 Phe into the recombinant proteins relies on activation of the photosensitive amino acid by the phenylalanyl-tRNA synthetase (PheRS) of the bacterial expression host. The PheRS used for this study was a previously characterized mutant with relaxed substrate specificity. 9 This method results in statistical decoding of phenylalanine (Phe) codons placed at regular intervals in the coding sequence. 9 Proteins were expressed in a Phe-auxotrophic E. coli strain and purified by exploiting the temperaturedependent phase behavior of proteins that contain elastin-like repeats. 10 Incorporation efficiency was determined by integration of the aromatic proton signals in the 1 H NMR spectra of the purified proteins; the extent of Phe replacement varied from 13% to 53%, depending on the concentration of pN 3 Phe in the expression medium (Supporting Information). Understanding the response of the photoreactive protein to irradiation is crucial for highresolution pattern formation....

Synthesis of proteins containing errors (mistranslation) is prevented by aminoacyl transfer RNA synthetases through their accurate aminoacylation of cognate tRNAs and their ability to correct occasional errors of aminoacylation by editing reactions. A principal source of mistranslation comes from mistaking glycine or serine for alanine, which can lead to serious cell and animal pathologies, including neurodegeneration. A single specific G.U base pair (G3.U70) marks a tRNA for aminoacylation by alanyl-tRNA synthetase. Mistranslation occurs when glycine or serine is joined to the G3.U70-containing tRNAs, and is prevented by the editing activity that clears the mischarged amino acid. Previously it was assumed that the specificity for recognition of tRNA(Ala) for editing was provided by the same structural determinants as used for aminoacylation. Here we show that the editing site of alanyl-tRNA synthetase, as an artificial recombinant fragment, targets mischarged tRNA(Ala) using a structural motif unrelated to that for aminoacylation so that, remarkably, two motifs (one for aminoacylation and one for editing) in the same enzyme independently can provide determinants for tRNA(Ala) recognition. The structural motif for editing is also found naturally in genome-encoded protein fragments that are widely distributed in evolution. These also recognize mischarged tRNA(Ala). Thus, through evolution, three different complexes with the same tRNA can guard against mistaking glycine or serine for alanine.

The unsaturated amino acid 2‐amino‐3‐methyl‐4‐pentenoic acid (E‐Ile) was prepared in the form of its (2S,3S),(2R,3R) and (2S,3R),(2R,3S) stereoisomeric pairs. The translational activities of SS‐E‐Ile and SR‐E‐Ile were assessed in an E. coli strain rendered auxotrophic for isoleucine. SS‐E‐Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72 %; SR‐E‐Ile yielded no conclusive evidence for incorporation. ATP/PPi exchange assays indicated that SS‐E‐Ile was activated by the isoleucyl‐tRNA synthetase at a rate comparable to that characteristic of isoleucine; SR‐E‐Ile was activated approximately 100‐times more slowly than SS‐E‐Ile.

Oxidation reactions are one of the main reasons for the failure of polymeric materials. Antioxidants, compounds designed to protect against oxidation, must meet three main requirements for success: (1) an efficient antioxidative mechanism, (2) compatibility with the oxidizing polymer, and (3) permanence within the oxidizing polymer. Common antioxidants are low molecular weight materials that can easily diffuse, leach, or evaporate from the polymer they are designed to protect. An increase in the molecular weight of the antioxidant not only decreases diffusion and volatility, but also decreases compatibility since most high molecular weight polymers will not mix. Selective sterically hindered phenolics, however, are concurrently antioxidants and "compatibilizers" through hydrogen bond formation. Carefully designed copolymers containing a small percentage of 2,6-diisopropyl-4-vinylphenol were found to mix intimately with two readily oxidizing polyethers and to protect them against oxidation.