We have biosynthetically incorporated several tryptophan analogues into three forms of Staphylococcal nuclease to investigate the spectroscopic characteristics of these "intrinsic" probes and their effect on the structure of the proteins. The set of tryptophan analogues includes 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan. 5-Hydroxytryptophan and 7-azatryptophan have red-shifted absorbance spectra, and the latter has a red-shifted fluorescence, which is very sensitive to its environment (being heavily quenched in water). The fluorotryptophans can serve as 19F NMR probes, and 4-fluorotryptophan has a very low fluorescence quantum yield, thus making it a "knock-out" fluorescence analogue. The set of proteins studied includes wild-type nuclease, which has a single tryptophan site at position 140; its V66W mutant, which has a second tryptophan at position 66; and the Delta 137-149 fragment, V66W', which only has a tryptophan at position 66. The environments of positions 66 and 140 are significantly different; position 140 is near the end of the long C-terminal alpha-helix and is moderately solvent-exposed, whereas position 66 is in the beta-barrel core region of the protein and is surrounded by apolar side chains. Absorbance and 19F NMR spectra are used to estimate the extent of analogue incorporation for each protein. Steady-state and time-resolved fluorescence data are reported to characterize the emission of the analogues in these positions in the three proteins and to develop the use of the analogues as probes of protein structure and dynamics. Circular dichroism spectra are reported to show that, in all but a couple of cases, the secondary structure of the proteins containing the analogues is not significantly perturbed by the probes. Additionally, fluorescence anisotropy decay data show the variants of wild-type nuclease to have a rotational correlation time similar to that of tryptophan-containing nuclease.
IIIGk is a signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system ofEscherichia coli. The secondary structure of EIIk is determined by heteronuclear (15N, 13C) three-dimensional NMR spectroscopy. Sequential, medium-range, and longrange nuclear Overhauser effects seen in NMR spectra are used to elucidate 11 antiparallel f-strands and four helical segments. The medium-range nuclear Overhauser effect patterns suggest that the helices are either distorted a-helices or are of the 310 class. The amino acids separating the active-site histidine residues (His75 and His") form two strands (Ala76-Ser8 and Val~s-Phe9l) of a six-stranded antiparallel fl-sheet that brings His9" and His75 in close proximity. Sequence similarities in EIic and several other sugar-transport proteins suggest that the histidine residues within these proteins may be arranged in a similar manner. The 18-residue N-terminal peptide that precedes f-strand Thr'9-le22 in native IGic is disordered and does not interact with the rest of the protein. Furthermore, removal of the N-terminal heptapeptide by a specific endopeptidase does not affect the structure of the remaining protein, thus explaining the phospho-acceptor activity of modified IHlk with the phospho-histidine-containing phosphocarrier protein of this system. The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) was first recognized as a sugar phosphorylating/transport system and subsequently as a key regulatory system for other processes. Many of the latter functions, such as diauxic growth, involve transduction of signals from the environment to the genome, thereby initiating or repressing the transcription of certain operons. The crr gene was found to be essential for these regulatory processes, and this gene was shown to encode the PTS phosphocarrier protein, IIIGIC (18.1 kDa). IIIGlc fulfills its many functions by interacting with membrane-bound and cytoplasmic proteins including the PTS proteins phosphohistidine-containing phosphocarrier protein of the PTS (HPr), enzyme 11GIc, enzyme IIBBg, and the non-PTS proteins adenylate cyclase, glycerol kinase, and the melibiose, lactose, and maltose permeases (for recent reviews, see refs. 1-3).Biochemical and physical methods have revealed several regions of 111GIc important for activity. HPr phosphorylates His' at the N-3 position (4), and replacement of this histidine residue with glutamine completely inactivates the protein. In contrast, replacement of His75 with glutamine results in the loss of phospho-donor but not phospho-acceptor activity, indicating that both histidine residues are required for normal function (5). Furthermore, cleavage of the N-terminal heptapeptide by a membrane-associated endopeptidase reduces phospho-donor activity by 97% (6), whereas derivitization of Gly' reduces this activity 80% (7). These results suggest that the N terminus is important in formation of the binary IIIGcI,_IIGlc complex.Early NMR investigations ofPTS proteins centered around ...
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.Keywords: 7-azatryptophan; CD studies of staphylococcal nuclease A; 5-hydroxytryptophan; nuclease A (staphylococcal); thermodynamics of unfolding; time-resolved fluorescence studies of staphylococcal nuclease A; tryptophan analogues There has been much interest in recent years in the strategy of incorporating unnatural amino acids into proteins, for example, as a means of specifically perturbing the chemical nature of a particular side chain to test for its contribution to the function of a protein.To cite a few examples, Schultz and coworkers have used a cell-free expression system (chemically attaching the desired unnatural amino acid to suppressor tRNA and then placing the amber codon at the desired position in the mRNA) to incorporate a number of unnatural amino acids, mostly aliphatic amino acid analogues, at various positions in T4 lysozyme (Noren et al., 1989;Ellman et al., 1992;Mendel et al., 1992) and staphylococcal nuclease (Judice et al., 1993;Thorson et al., 1995). The small amount of analogue-containing protein produced was then subjected to
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