SITE-SPECIFIC INCORPORATION OF BIOPHYSICAL PROBES INTO PROTEINS

Biophysical probes which can detect structural changes in proteins and the interaction of proteins with other macromolecules are important t ools in studying protein function. Many difficulties remain, however, in introducing probes into proteins site-specifically. Here we report the successful site-specific incorporation of a spin-labeled, a fluore scent, and a photoactivatible amino acid into a variety of surface and internal sites in bacteriophage T4 lysozyme by using unnatural amino acid mutagenesis. In addition, we report the purification and spectral characterization of T4 lysozyme mutants containing the spin-labeled a mino acid and the fluorescent amino acid. The ability to incorporate t hese probes site-specifically allows for novel studies of protein stru cture and dynamics. Moreover, this work demonstrates that the Escheric hia coli protein biosynthetic machinery can tolerate unnatural amino a cids with little resemblance to the natural amino acids.