Protein Science, Vol 6, Issue 3 689-697, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: Effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability

C. Y. WONG and M. R. EFTINK
Department of Chemistry, University of Mississippi, University, Mississippi 38677

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogues of tryptophan that potentially can be incorporated biosynthetically into proteins 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 that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor. Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate ~95% incorporation of 5HW into position 140 of nuclease, and we estimate ~98% incorporation of 7AW. CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.
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