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Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: Effect of 5‐hydroxytryptophan and 7‐azatryptophan on structure and stability
Author(s) -
Wong CingYuen,
Eftink Maurice R.
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060318
Subject(s) - tryptophan , nuclease , chemistry , guanidine , fluorescence , protein secondary structure , micrococcal nuclease , fluorescence anisotropy , tryptophan synthase , protein structure , stereochemistry , dna , biochemistry , amino acid , nucleosome , physics , quantum mechanics , membrane , histone
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 c 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.