We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-l-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.

Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase͞tRNA pair that makes possible the in vivo incorporation of p-benzoyl-L-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo.A ll organisms use the same common 20 amino acids as building blocks for the biosynthesis of proteins. The ability to augment the genetically encoded amino acids with unnatural amino acids containing orthogonal chemical handles, photocrosslinking groups, fluorescent probes, redox active groups, or heavy atoms would provide powerful tools for manipulating and probing protein function in vitro, in cells and perhaps in whole organisms. Recently, we reported that by adding new components to the translational machinery of Escherichia coli, additional amino acids could be site-specifically incorporated with high translational fidelity into proteins in vivo (1). We now show that this methodology can be used to site specifically incorporate photocrosslinking amino acids into a protein in E. coli.Benzophenones have been used extensively as photophysical probes to identify and map peptide-protein interactions (2). In contrast to aryl azides, diazoesters, and diazarenes, they are chemically stable and can be routinely manipulated under ambient light. Upon excitation at 350-360 nm, wavelengths that avoid protein damage, they preferentially react with otherwise unactivated carbon-hydrogen bonds (COH) (3, 4). Furthermore, in contrast to other photocrosslinking groups, benzophenones do not photodissociate and their photoexcited triplet state readily relaxes in the absence of a suitable COH bond with which to react. The reversible excitation of benzophenones allows repeated excitation by long-wavelength UV light and thus excellent crosslinking yields (5). Early uses of benzophenones included the functionalization of remote COH bonds in steroids; mapping the conformations of flexible chains in solution, micelles, and membranes (6, 7); and mapping the nucleotidebinding sites in ATPases (8).In 1986, DeGrado and coworkers (5) demonstrated that the photocrosslinking amino acid p-benzoyl-L-phenylalanine (pBpa) (Fig. 1A) could be site specifically incorporated into synthetic peptides by solid-phase peptide synthesis. Peptides incorporating this amino acid have been used extensively to probe peptideprotein interactions in numerous systems in vitro (2). These experiments have established that pBpa is a very efficient photocrosslinker, affording between 50% and 100% crosslinking between the peptides and their receptors. These experiments also demonstrated that the crosslinks are formed selectively between the benzophenone moiety and ...

We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.