BIOSYNTHETIC INCORPORATION OF TRYPTOPHAN ANALOGS INTO STAPHYLOCOCCAL NUCLEASE - EFFECT OF 5-HYDROXYTRYPTOPHAN AND 7-AZATRYPTOPHAN ON STRUCTURE AND STABILITY

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogues of t ryptophan that potentially can be incorporated biosynthetically into p roteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration tha t they cause minimal perturbation of a protein's structure and stabili ty. To investigate these factors in a model protein, we have incorpora ted 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the tem perature-sensitive lambda cI repressor. Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of a bsorption spectra, we estimate similar to 95% incorporation of 5HW int o position 140 of nuclease, and we estimate similar to 98% incorporati on of 7AW. CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secon dary structure is not changed by the tryptophan analogues. Steady-stat e fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluores cence intensity and anisotropy measurements indicate that the incorpor ated 5HW residue, like tryptophan at position 140, has a dominant rota tional correlation time that is approximately the value expected for g lobal rotation of the protein. Guanidine-hydrochloride-induced unfoldi ng studies show the unfolding transition to be two-state for 5HW-conta ining protein, with a free energy change for unfolding that is equal t o that of the tryptophan-containing protein. In contrast, the guanidin e-hydrochloride-induced unfolding of 7AW-containing nuclease appears t o show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.