©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand-mediated Changes in the Tryptophan Synthase Indole Tunnel Probed by Nile Red Fluorescence with Wild Type, Mutant, and Chemically Modified Enzymes (*)

(Received for publication, November 22, 1994; and in revised form, January 12, 1995)

Sergei B. Ruvinov (1) Xiang-Jiao Yang (1) Kevin D. Parris (2) Utpal Banik (1) S. Ashraf Ahmed (1) Edith Wilson Miles (1)(§) Dan L. Sackett (1)(§)

From the  (1)Laboratory of Biochemical Pharmacology and (2)Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The bacterial tryptophan synthase alpha(2)beta(2) complex contains an unusual structural feature: an intramolecular tunnel that channels indole from the active site of the alpha subunit to the active site of the beta subunit 25 Å away. Here we investigate the role of the tunnel in communication between the alpha and beta subunits using the polarity-sensitive fluorescent probe, Nile Red. Interaction of Nile Red in the nonpolar tunnel near beta subunit residues Cys-170 and Phe-280 is supported by studies with enzymes altered at these positions. Restricting the tunnel by enlarging Cys-170 by chemical modification or mutagenesis decreases the fluorescence of Nile Red by 30-70%. Removal of a partial restriction in the tunnel by replacing Phe-280 by Cys or Ser increases the fluorescence of Nile Red more than 2-fold. A binding site for Nile Red in this region near the pyridoxal phosphate coenzyme of the beta subunit is further supported by iodide quenching and fluorescence energy transfer experiments and by molecular modeling based on the three-dimensional structure of the alpha(2)beta(2) complex. Finally, studies using Nile Red as a sensitive probe of conformational changes in the tunnel reveal that allosteric ligands (alpha subunit) or active site ligands (beta subunit) decrease the fluorescence of Nile Red. We speculate that allosteric and active site ligands induce a tunnel restriction near Phe-280 that serves as a gate to control passage of indole through the tunnel.


INTRODUCTION

Metabolite channeling, the subject of considerable recent interest and some controversy(1, 2) , is the process by which the product of an enzymatic reaction is passed directly to the next enzyme in a biosynthetic pathway rather than by diffusion through solution. Tryptophan synthase (EC 4.2.1.20), a classic enzyme that exhibits metabolite channeling, catalyzes the last two steps in the biosynthesis of L-tryptophan. The bacterial enzyme is an alpha(2)beta(2) complex that dissociates reversibly into monomeric alpha subunits and a beta(2) dimer (for reviews, see (3, 4, 5, 6, 7) ). The alpha subunit catalyzes cleavage of indole-3-glycerol phosphate to indole and D-glyceraldehyde 3-phosphate, termed the alpha reaction. The beta subunit catalyzes the pyridoxal phosphate-dependent condensation of indole with L-serine to form L-tryptophan, termed the beta reaction. The physiologically important reaction catalyzed by the alpha(2)beta(2) complex, termed the alphabeta reaction, is the sum of the alpha and beta reactions (Fig. SIA). The finding that indole does not appear as a free intermediate in solution in the alphabeta reaction provides evidence that indole passes intramolecularly from the alpha site to the beta site(8, 9, 10) . Channeling of indole is supported by rapid kinetic experiments(11, 12, 13, 14) . The crystal structure of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium(15) has provided a plausible structural basis for channeling by revealing a 25-Å tunnel that connects the catalytic sites of the alpha and beta subunits.


Scheme I: A, in the tryptophan synthase alphabeta reaction indole-3-glycerol phosphate (IGP) is cleaved to D-glyceraldehyde 3-phosphate (G-3-P) and an indole intermediate ([IND]) at the alpha site. The indole intermediate diffuses through a 25-30 Å tunnel to the beta site where it undergoes a pyridoxal phosphate (PLP)-dependent reaction with L-Ser to form L-Trp. B, active site of the beta subunit based on the x-ray crystallographic results(15) . The figure, which was prepared by C. C, Hyde, shows cysteine 170, phenylalanine 280, the pyridoxal phosphate:Lys-87 Schiff base, indole positioned near the end of a tunnel that extends from the alpha site to the beta site, and the approximate surface of the tunnel (dots). The dashed side chains show the positions of phenylalanine 170 created by computer graphics modeling and an alternative position of phenylalanine 280 seen in some crystal structures (see text). The surface of the tunnel, shown by dots, has been described(15) . The alpha/beta interaction site is located to the left of region shown. This site and pyridoxal phosphate are approximately equidistant from Cys-170.



Fig. SIB shows part of the tunnel within the beta subunit, the Schiff base formed between pyridoxal phosphate and the -amino group of lysine 87 at the active site of the beta subunit, and two residues in the wall of the tunnel, Cys-170 and Phe-280. Molecular modeling suggests that a bulky side chain at position 170 might restrict the tunnel and interact with the aromatic side chain of Phe-280, a residue on the opposite wall of the tunnel that appears to enter the tunnel in some crystal structures. (^1)The alternative position of the chain of Phe-280 is shown by a dashed line in Fig. SIB. Furthermore, substitution of Phe-280 by a less bulky residue might prevent restriction of the tunnel at this position. Fig. SIB also shows an indole molecule positioned by computer graphics modeling at a likely position for reaction with the L-serine-pyridoxal phosphate intermediate to form L-tryptophan.

In the present work, we have followed the suggestions of molecular modeling described above to construct several specifically altered forms of the alpha(2)beta(2) complex. To increase the bulk of beta subunit Cys-170, we have used chemical modification by N-ethylmaleimide to prepare C170-NEM (^2)and by methyl methanethiolsulfonate to make C170-MMTS. We have also used site-directed mutagenesis to replace Cys-170 by phenylalanine (C170F) and by tryptophan (C170W). Recent crystallographic and kinetic analyses of the C170W mutant form of the alpha(2)beta(2) complex prepared as described here for the first time show that the engineered tryptophan side chain does indeed obstruct the tunnel and results in the transient accumulation of an indole intermediate(16) . To reduce the partial restriction of the tunnel by Phe-280, we have replaced Phe-280 by cysteine (F280C) and by serine (F280S). The several new forms of the alpha(2)beta(2) complex engineered in the present work should provide useful materials for future studies of the kinetics of indole channeling.

Because residues that form the surface of the tunnel are largely nonpolar(15) , nonpolar compounds other than indole may bind in the tunnel and serve as probes of tunnel structure and function. In the present work, we investigate the interaction of the polarity-sensitive fluorescent dye, Nile Red(17, 18, 19) , with various forms of wild type tryptophan synthase and with alpha(2)beta(2) complexes having alterations in the tunnel. Our results provide evidence that Nile Red binds in the tunnel near the active site of the beta subunit and that the geometry of this region of the tunnel is altered by beta site ligands and by allosteric alpha site ligands. These ligands may induce a tunnel restriction near Phe-280 that serves as a gate to control passage of indole through the tunnel.


MATERIALS AND METHODS

Chemicals and Buffers

Pyridoxal phosphate, DL-alpha-glycerol 3-phosphate, EDTA, 5,5`-dithiobis(2-nitrobenzoic acid), amino acids, indole, and benzimidazole were from Sigma. N-Ethylmaleimide was from Fluka and methyl methanethiolsulfonate from Aldrich. Buffer B (50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8) was used for all spectroscopic studies. Buffer P (0.1 M potassium phosphate, pH 7.8, containing 1 mM EDTA, 0.45 M (NH(4))(2)SO(4), and 0.1 mM pyridoxal phosphate) was used for sulfhydryl modification. Indole-3-glycerol phosphate was synthesized enzymatically and purified as described(20) Indole-3-propanol phosphate was a generous gift of Kasper Kirschner(21) . Nile Red, 9-diethylamino-5H-benzo[alpha] phenoxazine-5-one, was obtained from Eastman Kodak Co. and was used without further purification. Stock solutions of Nile Red were prepared in dimethyl sulfoxide (Me(2)SO) or dimethylformamide and stored at -20 °C.

Determination of Nile Red Solubility

To determine the solubility of Nile Red in Buffer B, an aliquot of a concentrated stock solution of Nile Red in dimethylformamide (2.1 mM) was diluted to 0.7 ml with Buffer B in a microcentrifuge tube to a final concentration of 5 µM. After 30 min the tube was centrifuged for 5 min at top speed in a microcentrifuge to precipitate insoluble aggregates of Nile Red. The top 0.3 ml was carefully removed, transferred to a microcentrifuge tube, and extracted with 0.3 ml of dichloroethane by vigorous vortex mixing for 15 s followed by centrifugation for 1 min to separate the phases. The pink organic layer was removed and the absorbance measured at 538 nm. The concentration of Nile Red was calculated to be 0.2 µM using a molar extinction coefficient of 4 times 10^4M cm (Kodak Chemical Co.; Molecular Probes). To avoid complications due to formation of a third component of insoluble Nile Red in addition to the enzyme-bound and free soluble forms as occurs in aqueous solution of cholesterol(22) , we have limited the concentration of added Nile Red to 1 µM in most studies in this paper. Therefore, the dye is always substoichiometric to the enzyme (5 µM).

Enzymes and Enzyme Assays

Wild type tryptophan synthase alpha and beta subunits from S. typhimurium were purified from extracts of the Escherichia coli (CB 149) host that harbors high copy plasmids carrying the wild type trpA or trpB genes from S. typhimurium, respectively (23) . Wild type or mutant forms of the tryptophan synthase holo-alpha(2)beta(2) complex from S. typhimurium were purified and crystallized from a host strain of E. coli (CB 149) that harbors a high copy plasmid carrying trpA and trpB genes from S. typhimurium(24) . Plasmid pSTB7(20) (^3)was used for expression of the wild type alpha(2)beta(2) complex. The mutated derivatives of phagemid pTZ52 (25) (see below) were used for expression of the mutant alpha(2)beta(2) complexes. The purity of each enzyme preparation was established to be greater than 95% by sodium dodecyl sulfate-gel electrophoresis on 10-15% gradient gels using a Phastgel System (Pharmacia Biotech Inc). The reduced alpha(2)beta(2) complex was prepared by treatment of the holo-alpha(2)beta(2) complex in Buffer B with sodium borohydride followed by dialysis(26) . Apo-beta subunit and apo-alpha(2)beta(2) complex were prepared from the corresponding holoenzymes as described(27, 28) . Tryptic cleavage of the alpha subunit loop in the alpha(2)beta(2) complex was carried out with 5 µg/ml TPCK-trypsin (Cooper Biochemical) for 10 min at room temperature and terminated by addition of soybean trypsin inhibitor as described(29, 30) . Cleavage of the separate alpha subunit was carried out in the same way using 1 µg/ml TPCK-trypsin for 10 min. The effects of proteolysis were checked by sodium dodecyl sulfate electrophoresis and staining with Coomassie Blue R-350 utilizing a Phast System (Pharmacia Biotech Inc.)(30) . The results showed the presence of two protein bands (alpha-1 and alpha-2) in the nicked alpha subunit and three protein bands (alpha-1 and alpha-2 and beta) in the nicked alpha(2)beta(2) complex. Protein concentrations were determined from the specific absorbance at 278 nm using A values 4.4 for the alpha subunit, 6.5 for the holo-beta subunit, 5.8 for the apo-beta subunit and 6.0 for the holo-alpha(2)beta(2) complex, respectively(31) . Enzyme activity in the alphabeta reaction (L-serine + indole-3-glycerol phosphate L-tryptophan + D-glyceraldehyde 3-phosphate) was measured by a spectrophotometric assay coupled with D-glyceraldehyde-3-phosphate dehydrogenase (Sigma) (32) and in the beta reaction (L-serine + indole L-tryptophan) by a direct spectrophotometric assay(31) . One unit of activity is the formation of 0.1 µmol of product in 20 min at 37 °C. The specific activities of the purified wild type and mutant (see below) holo-alpha(2)beta(2) complexes in the beta reaction were wild type (1000 units/mg), C170F (200 units/mg), C170W (40 units/mg), F280S (510 units/mg), F280C (780 units/mg).

Vector Constructions and Oligonucleotide-directed Mutagenesis

Standard cloning methods were used(33) . Plasmid transformation was performed using a published procedure(34) . Phagemid pTZ52 was used as a convenient vector for mutagenesis and for expression of the tryptophan synthase alpha(2)beta(2) complex(25) . Oligonucleotide-directed mutagenesis utilized single-stranded uracil-rich DNA from CJ236 (35) harboring pTZ52 superinfected by helper phage M13K07 (Bio-Rad) or R408(36) . The trpB phenylalanine 280 codon was changed to the codon for serine or cysteine using mutagenic oligomers (21-mers) 5`-TTT CAT CCC G GA ATA GAT GCC-3` or 5`-TTT CAT CCC G CA ATA GAT GCC-3`, respectively, where the mutated nucleotide is underlined. The trpB cysteine 170 codon was changed to the codon for phenylalanine or tryptophan using mutagenic oligomers (21-mers) 5`-CGC CTC GTT AA*A GGC ATC TTT-3`or 5`-CGC CTC GTT C*CA GA*G* ATC TTT-3`, respectively. The asterisk follows the base changed, and altered codons are underlined. Additional changes were made in the second oligonucleotide to introduce a BglII site to facilitate screening. The new BglII site is potentially useful for cassette mutagenesis. The last oligonucleotide introduces a second amino acid change: A169L. Although the amount of single-stranded DNA obtained by superinfection was sufficient for mutagenesis, it was insufficient for sequencing. Therefore, the desired mutations were confirmed by dideoxy DNA sequencing of the double-stranded plasmid DNA using a Sequenase(TM) kit (U. S. Biochemical Corp).

Spectroscopic Methods

Absorption spectra were measured in a Hewlett-Packard 8452 diode array spectrophotometer at room temperature. Steady-state fluorescence measurements were made with a Perkin-Elmer MPF-66 spectrofluorimeter in ratio mode, equipped with a red-sensitive Hamamatsu R928 photomultiplier. Microcells with a working volume of 250 µl and a path length of 5 mm (NSG Precision Cells, Farmingdale, NY) were used throughout. Spectra were collected using 5-nm excitation and emission slits. Data were collected with a dedicated Perkin-Elmer Series 7300 professional computer, and spectral analysis was performed with a software package from Perkin-Elmer (PECLS-III, Perkin-Elmer computerized luminescence software package). Nile Red was added to protein solutions in Buffer B to the desired final concentrations from stocks such that the addition was less than 1% of the final volume. These working solutions were prepared by dilution in dimethyl sulfoxide of a single stock of 2 mM in dimethyl sulfoxide. Spectra were recorded 15 min after addition of Nile Red and are uncorrected. This period minimized time-dependent changes, which facilitated comparison of spectra(18) . Excitation was at 550 nm unless otherwise indicated. All experiments and measurements were carried out at room temperature. Fluorescence lifetimes were obtained from time-resolved measurements made with a single photon counting instrument described in detail elsewhere(37) . The fluorescence lifetimes of Nile Red in dimethylformamide and in the nonpolar site in the alpha(2)beta(2) complex were obtained from decay-associated spectra, collected as described(19, 38) .

Data Analysis

Spectral components in steady-state fluorescence spectra were deconvoluted using the nonlinear least squares fitting routines of PeakFit software (Jandel Scientific, Corte Madera, CA). Spectra were fit as sums of Gaussians following conversion to intensity versus wavenumber. All spectra were fit with two components: the protein bound component, fit as a Gaussian whose center, amplitude, and bandwidth were fitting parameters, and an aqueous component. The aqueous component was first fit using Nile Red in Buffer B alone. The parameters of this fit (except for amplitude) were held constant when fitting spectra with emission due to protein bound dye. The spectrum of Nile Red in Buffer B alone could not be fit as a Gaussian or as a sum of two Gaussians, but could be fit to a Voigt function with a small Gaussian addition. The good fit achieved with the Voigt is likely due to collisional broadening of Nile Red emission in aqueous solution(39) . The origin of the small Gaussian component, which has been observed in a variety of solvents, is not clear, but may be due to a second transition of lower energy.

Iodide Quenching of Fluorescence of Nile Red and Pyridoxal Phosphate

Fluorescence emission spectra with excitation at 550 nm were recorded from samples of Nile Red bound to various forms of tryptophan synthase or dissolved in pure solvent. Fluorescence emission spectra with excitation at 410 nm were recorded from samples of pyridoxal phosphate bound to various forms of tryptophan synthase. Small additions were made of a stock solution of potassium iodide in Buffer B, and the emission spectra were recorded after each addition. Sodium thiosulfate (0.01 mM) was added to the iodide stock to avoid oxidation. Since high concentrations of iodide cause dissociation of the alpha(2)beta(2) complex(27, 40) , the quenching analysis used final iodide concentrations less than 0.3 M. Fluorescence emission intensity at 600 nm was noted from the recorded spectra in order to monitor emission from the nonpolar site of the enzyme and avoid a contribution from the aqueous component. The fluorescence emission at 620 nm was monitored for a solution of Nile Red in dimethylformamide. Quenching constants were obtained from Stern-Volmer plots, which were linear in the range examined.

Energy Transfer Measurements

The distance between two fluorophores with appropriate properties may be estimated from measurements of resonance energy transfer according to the theory of Förster (reviewed in (41) ). One requirement for this approach is that the emission spectrum of one fluorophore (the donor) overlap the absorbance (excitation) spectrum of the second (the acceptor). This applies to pyridoxal phosphate (or reduced pyridoxal phosphate) and Nile Red. Under this situation the efficiency of energy transfer from donor to acceptor, E, is related to the distance R between donor and acceptor as shown the following equation.

R(o) is the Forster critical distance at which transfer of excitation energy is 50%. R(o) is defined (in Angstroms) by,

where n is the refractive index, taken as 1.4, (D) is the donor quantum yield, taken to be 0.018 for pyridoxal phosphate (40) and 0.027 for reduced pyridoxal phosphate, kappa^2 is an orientation factor that can have values between 0 and 4 and is two-thirds if the fluorophores can freely rotate, and J is the spectral overlap integral. J was evaluated numerically using 10-nm interval values from the normalized emission spectra of pyridoxal phosphate (or reduced pyridoxal phosphate) and the absorbance spectrum of Nile Red in 80% dioxane/water, a solvent in which Nile Red emission has properties close to those of the enzyme-bound dye.

The efficiency of excitation transfer from donor to acceptor, E, is defined as follows,

where F(D) is the fluorescence of the donor in the absence of acceptor and F is the corresponding donor fluorescence in the presence of saturating acceptor. Since the solubility of Nile Red in aqueous solution prevented achievement of saturation, the observed transfer efficiency must be corrected for the fraction bound. This was done by extrapolation. Emission spectra of pyridoxal phosphate or reduced pyridoxal phosphate were recorded in the absence of Nile Red and in the presence of varying Nile Red concentrations up to 1 µM, corresponding to a free dye concentration of about 0.2 µM based on the determined K(d) of 1 µM. The reduction in integrated intensity (DeltaF) of emission was determined for each concentration of Nile Red. The value of DeltaF at saturation was estimated from the intercept of the data plotted in double-reciprocal format (42; see also (43) ),

where B is a constant and [L(f)] is the concentration of free ligand, here Nile Red.

The evaluation of spectral overlap integrals yielded an overlap integral value of J = 19 times 10^14M cm nm^4 for pyridoxal phosphate and Nile Red and a value of J = 1.1 times 10^14M cm nm^4 for reduced pyridoxal phosphate and Nile Red. These values correspond to critical Forster distances (R(o)) of 28 and 19 Å, respectively, assuming rotational averaging of the fluorophores' emission. Orientation effects may not be trivial, as steady-state and time-resolved measurements indicate significant anisotropy for the Nile Red with the alpha(2)beta(2) complex compared with Nile Red in solution (data not shown). If the assumption of rotational averaging in the donor-acceptor pair is not valid, the value of R(o) for the reduced pyridoxal phosphate-Nile Red pair might have the range of 8-22 Å.

Molecular Modeling

The Nile Red molecule was first built using Chem Draw (Cambridge Scientific Computing, Inc.) on a Macintosh II. The coordinates were then imported into QUANTA (Molecular Simulation, Inc.) on a Silicon Graphics 4D310 and minimized energetically using the CHARMm module. The solvent-accessible surface of the wild type tryptophan synthase alpha(2)beta(2) complex was calculated using a suite of programs (MS)(44) . The dot surface generated by this program was then imported into PSFrodo (45) on an Evans and Sutherland PS390 along with the energy minimized structure of Nile Red. The van der Waal surface of Nile Red was then generated using the SURFACE command within PSFrodo. The position of Nile Red was then manipulated by hand as described in the text.

Chemical Modification of beta Subunit Cys-170

Treatment of the holo-beta subunit from E. coli with N-ethylmaleimide results in the specific modification of one of the five sulfhydryl residues (46) later shown to be Cys-170(47) . We have used this procedure to modify the holo-beta subunit from S. typhimurium. The holo-beta subunit was activated by treatment for 30 min at 37 °C in Buffer B containing 0.1 mM pyridoxal phosphate and 2 mM dithiothreitol and then converted into Buffer P by gel filtration on a PD-10 column. Holo-beta subunit (0.1 mM in Buffer P) was treated with 0.25 mMN-ethylmaleimide for 30 min at room temperature followed by addition of 2.5 mM 2-mercaptoethanol and dialysis versus Buffer B, as described (47) or with 0.25 mM methyl methanethiolsulfonate (48) for 30 min at room temperature followed by gel filtration in Buffer B. Holo-beta subunits modified by N-ethylmaleimide or methyl methanethiolsulfonate were designated C170-NEM or C170-MMTS respectively. Treatment of the modified and untreated beta subunits with 5,5`-dithiobis(2-nitrobenzoic acid) was used to determine the number of reactive sulfhydryl groups in Buffer B and the number of total sulfhydryl groups in Buffer B containing 5 M urea(49) . The results demonstrate that the single reactive sulfhydryl in the holo-beta subunit was modified by each reagent (data not shown) as shown previously (46) after the reaction of N-ethylmaleimide with holo-beta subunit from E. coli. Titration of the activities of the untreated beta subunit, beta-C170-NEM and beta-Cys-170-MMTS with alpha subunit (data not shown) demonstrated that each beta subunit forms a complex with approximately one alpha subunit as shown previously (46) after the reaction of N-ethylmaleimide with holo-beta subunit from E. coli. The modified alpha(2)beta(2) complexes were prepared by mixing equimolar alpha and beta subunits. The activities of the beta-C170-NEM and beta-Cys-170-MMTS alpha(2)beta(2) complexes were about 25 and 50%, respectively, that of the untreated alpha(2)beta(2) complex in the beta reaction.


RESULTS

To define functional roles of the tunnel that connects the active sites of tryptophan synthase alpha and beta subunits (Fig. SI), we have engineered alterations in beta subunit residues Cys-170 and Phe-280 by site-directed mutagenesis and chemical modification (see the Introduction and ``Methods and Materials''). We have compared the interaction of the wild type and altered forms of the tryptophan synthase alpha(2)beta(2) complex with the polarity sensitive dye Nile Red to obtain evidence for its site of interaction. Finally, we have analyzed the effects of active site (beta subunit) and allosteric ligands (alpha subunit) on the interaction of Nile Red with the alpha(2)beta(2) complex.

Interaction of Nile Red with Tryptophan Synthase

The fluorescence properties of Nile Red, an uncharged phenoxazone dye with structure shown in Fig. 1A, are altered by the effective polarity of the environment. The dye is a sensitive probe of nonpolar surfaces of proteins and can reveal differences between alternate structural states(17, 18, 19, 50) . The fluorescence emission spectra of Nile Red (with excitation at 550 nm) in the presence of the tryptophan synthase alpha subunit, holo-beta subunit, and holo-alpha(2)beta(2) complex (Fig. 1, A-C) are complex, reflecting the presence of more than one environment for Nile Red. Deconvolution analysis (see ``Materials and Methods'') shows the presence of two main emission components in each fluorescence spectrum. In the case of the holo-beta subunit and holo-alpha(2)beta(2) complex, one component has a maximum near 625 nm, which is characteristic of a nonpolar environment with properties comparable with octanol, 2-propanol, or dimethylformamide(18) . We will refer to this as the nonpolar component. The other component has a maximum near 665 nm, which is characteristic of emission of Nile Red in an aqueous environment (19) and that from Nile Red in buffer alone. Table 1shows the deconvolution analysis of the emission spectra in Fig. 1. This analysis demonstrates the similarity of interaction of Nile Red with the holo-beta subunit and with the holo-alpha(2)beta(2) complex. That is, the nonpolar components for these forms have closely similar wavelength maxima (625-627 nm) and integrated emission intensities (8.2-8.7). In contrast, the nonpolar component for the alpha subunit has a lower wavelength maximum (619 nm) and a lower integrated intensity (3.2). The similarity of the spectra in Fig. 1, B and C, is partial evidence that Nile Red interacts with the same nonpolar site in the holo-beta subunit and holo-alpha(2)beta(2) complex. Furthermore, since the spectra in Fig. 1C are clearly not the sum of the spectra in Fig. 1, A and B (see legend, Fig. 1), the sites that interact with Nile Red in the separate alpha subunit do not appear to be available in the holo-alpha(2)beta(2) complex.


Figure 1: Emission spectra of Nile Red in the presence of the tryptophan synthase alpha and holo-beta subunits and holo-alpha(2)beta(2) complex. The structure of Nile Red is given in A. Proteins (5 µM protomer in Buffer B) were mixed with Nile Red (1 µM). Spectra were taken after 15 min at room temperature and were deconvoluted as described under ``Material and Methods.'' A, alpha subunit; B, holo-beta subunit; C, holo-alpha(2)beta(2). Each panel shows the total emission and the components revealed by deconvolution. The aqueous component is characterized by higher wavelength emission. The non-aqueous component emits at lower wavelength and is referred to in the text and Table 1and Table 3as the ``nonpolar component.'' Note that the total intensities and nonpolar components of curves for the holo-beta subunits in B are similar to the intensities of the corresponding curves for the holo-alpha(2)beta(2) complex in C. Details of the total and nonpolar component emission are given in Table 1for different forms of the enzyme.







Interaction of Nile Red with Altered Forms of the Holo-alpha(2)beta(2) Complex

To obtain evidence for the site of interaction of Nile Red, we have compared the fluorescence emission of Nile Red with the wild type alpha(2)beta(2) complex and with complexes having engineered alterations of beta subunit Cys-170 (Fig. 2A and Table 1) and Phe-280 (Fig. 2B and Table 1). Modification of Cys-170 by N-ethylmaleimide alters the fluorescence spectrum and results in a 30% decrease in the nonpolar component of emission. In contrast, modification by methyl methanethiolsulfonate, which introduces a smaller -SCH(3) group, has little effect on the fluorescence spectrum and the emission components. Replacing Cys-170 by a more bulky phenylalanine or tryptophan residue decreases the nonpolar emission of Nile Red by 30% (C170F) or 70% (C170W). Replacing beta subunit Phe-280 by a less bulky cysteine (F280C) or serine (F280S) residue greatly enhances the fluorescence emission of Nile Red (Fig. 2B), changes the shape of the spectra (Fig. 2B), and increases the nonpolar emission of Nile Red more than 2-fold (Table 1). Our finding that increasing the size of beta subunit residue 170 decreases the fluorescence of Nile Red (Fig. 2A), whereas decreasing the size of beta subunit residue 280 increases the fluorescence of Nile Red (Fig. 2B) suggests that Nile Red binds in a region of the tunnel near beta subunit residues 280 and 170 (see Fig. SIB). These residues are 16-18 Å from the pyridoxal phosphate coenzyme.



Figure 2: Emission spectra of Nile Red in the presence of wild type and altered forms of the tryptophan synthase holo-alpha(2)beta(2) complex. A, wild type holo-alpha(2)beta(2) complex and holo-alpha(2)beta(2) complexes with modifications at beta subunit Cys-170 (C170-MMTS, C170-NEM, C170F, and C170W). B, wild type holo-alpha(2)beta(2) complex and holo-alpha(2)beta(2) complexes with modifications at beta subunit Phe-280 (F280S and F280C). Details of the total emission and nonpolar components are given in Table 1for different forms of the enzyme.



Effect of Nile Red Concentration on Fluorescence Emission

Titration of the holo-alpha(2)beta(2) complex (5 µM) with Nile Red (0.2-2 µM) yields a family of emission spectra (Fig. 3A). At low concentrations of added probe, the emission maximum (625 nm) is near that of the nonpolar component in Table 1. Increasing the concentration of Nile Red increases the intensity and results in an apparent red shift of the center of the peak. The shift is due to an increase in the proportion of the aqueous component with maximum fluorescence at 665 nm, rather than to a shift in the character of the components. This shift is seen clearly in the spectra at 0.2 and 2 µM Nile Red normalized to maximum intensity (Fig. 3B). Deconvolution of the spectra yields the nonpolar component due to protein binding. Analysis of the binding data over a range of 0.1-1.25 µM by the plot in Fig. 3C labeled ``no ligand'' indicates a single site with K(d) of about 1.0 µM. Thus, the enzyme is not saturated by Nile Red at the highest concentration used. We were unable to use higher concentrations of Nile Red, because the solubility of free Nile Red in aqueous solutions is quite low (0.2 µM, see ``Materials and Methods''). In subsequent experiments, Nile Red was used at less than 1 µM yielding free concentration of 0.2 µM. The titration data complement the spectral deconvolution results, which indicate one class of sites per holo-alpha(2)beta(2) complex. Binding data in Fig. 3C in the presence of D-tryptophan and indole are described in a later section.


Figure 3: Titration of the holo-alpha(2)beta(2) complex with Nile Red. A, holo-alpha(2)beta(2) complex (5 µM) was treated with Nile Red (0.2 to 2 µM) for 15 min before recording of the emission spectra. B, curves from A with 0.2 and 2 µM Nile Red are normalized to the same maximum intensity. C, the reciprocal integrated emission intensity of the nonpolar component derived from the deconvolution of each curve in A and analogous curves collected in the presence of ligands is plotted versus the reciprocal concentration of Nile Red. circle, no ligand; box, 4 mM indole; up triangle, 1 mMD-tryptophan.



Nile Red Does Not Inhibit Enzymatic Activity

The activity of the holo-alpha(2)beta(2) complex in the conversion of L-serine and indole-3-glycerol phosphate to D-glyceraldehyde 3-phosphate and L-tryptophan was determined as described under ``Materials and Methods.'' The enzyme (0.01 µM) was preincubated with Nile Red (0.2 µM) in the reaction mixture for 15 min prior to addition of indole-3-glycerol phosphate and L-serine to start the reaction. Preincubation with Nile Red had no effect on the reaction rate. We would have expected a maximum inhibition of 17% based on the measured K(d) = 1 µM for Nile Red.

Iodide Quenching of the Fluorescence of Nile Red and of Pyridoxal Phosphate

To probe the solvent accessibility of Nile Red in its nonpolar binding site in the holo-alpha(2)beta(2) complex, we have examined iodide quenching of Nile Red fluorescence with the wild type enzyme and with two mutant forms (F280S and F280C) (Fig. 4B and Table 2) and compared this with quenching of pyridoxal phosphate fluorescence in the same forms of the alpha(2)beta(2) complex (Fig. 4A and Table 2). Before beginning this study, we needed to validate iodide as a quencher of Nile Red fluorescence, since another commonly used quencher, acrylamide, does not quench Nile Red in pure solvent(19) . In contrast (Table 2), iodide is an efficient quencher of Nile Red in dimethylformamide, a solvent chosen for its similarity (Nile Red emission maximum = 625 nm) to the environment of the protein-bound probe.


Figure 4: Iodide quenching of the fluorescence of pyridoxal phosphate (A) and Nile Red (B) bound to the wild type, F280C, and F280S holo-alpha(2)beta(2) complexes. See ``Materials and Methods.'' bullet, wild type; , F280C; , F280S.





Table 2presents the results of iodide quenching of Nile Red bound to the wild type holo-beta subunit and to the wild type and F280S and F280C holo-alpha(2)beta(2) complexes. Emission from Nile Red bound to the alpha subunit could not be evaluated, since the presence of even 50-100 mM KI apparently destabilized the protein and altered the Nile Red spectrum (data not shown). However, alpha subunit that was cleaved at Arg-188 by trypsin (51) (``nicked'' alpha subunit; see ``Materials and Methods'') was not destabilized by KI. Thus iodide quenching results with the nicked alpha subunit are presented in Table 2. Fluorescence of Nile Red bound to nicked alpha subunit was poorly quenched by KI, exhibiting a Stern-Volmer constant (K) of <1 M. It is noteworthy that the fluorescence of indole-3-propanol phosphate bound to the alpha subunit is also poorly quenched (by acrylamide)(40) . KI did quench Nile Red emission from the holo-beta subunit or holo-alpha(2)beta(2) complex without destabilizing these enzymes. The K values obtained with the holo-beta subunit and holo-alpha(2)beta(2) complex were similar and were larger than that obtained with the nicked alpha subunit, supporting the view that Nile Red is bound to beta in the alpha(2)beta(2) complex.

The K values for Nile Red obtained with the F280C and F280S alpha(2)beta(2) complexes were much higher than that for the wild type holo-alpha(2)beta(2) complex (Table 2). Comparison with quenching of pyridoxal phosphate showed that quenching constants for both Nile Red and pyridoxal phosphate were in the order: wild type < F280C F280S ( Table 2and Fig. 4), indicating that the mutations affected the environments of the pyridoxal phosphate and Nile Red similarly, consistent with the suggestion that the two fluorophores are close together.

To determine the mechanism of KI quenching, we obtained fluorescence lifetimes of the Nile Red-alpha(2)beta(2) complex in the absence or presence of KI. Surprisingly, the lifetimes were nearly unchanged by sufficient KI to yield 30-50% quench (data not shown). This was true for the wild type enzyme and for the F280S and F280C enzymes, for which the K values were much higher. Thus in all cases, the mechanism of quenching is static, not collisional, since this would result in parallel decrease in intensity and lifetime, as observed in dimethylformamide.

Since the quenching is not collisional, the extent of shielding of the bound Nile Red from free solvent cannot be evaluated quantitatively. (^4)However, the results do indicate that the bound Nile Red is protected from free contact with KI, since the quenching in dimethylformamide proves that iodide is an efficient collisonal quencher, if it can contact the Nile Red. In addition, the similar responses of KI quenching of Nile Red and pyridoxal phosphate to alteration of the Phe-280 residue suggest proximity of the fluorophores.

Fluorescence Energy Transfer from Pyridoxal Phosphate to Nile Red

Studies of the effects of modification of Cys-170 (see above and Fig. 2A and Table 1) indicate that Nile Red binds near Cys-170 and hence relatively near pyridoxal phosphate. The SH group of Cys-170 is about 16 Å from the center of the pyridoxal ring (see Fig. SIB). Such proximity might allow non-radiative energy transfer to occur from pyridoxal phosphate to Nile Red and permit estimation of a separation distance. Resonance energy transfer of excitation energy requires that the emission spectrum of the donor (lower wavelength) molecule overlap with the excitation spectrum of the acceptor and may be detected by the decrease in emission of the donor in the presence of bound acceptor. The spectral requirements are well met by pyridoxal phosphate or reduced pyridoxal phosphate and Nile Red. Spectral overlap integrals for these two pairs were evaluated as described under ``Materials and Methods.''

The data in Fig. 5demonstrate transfer of excitation energy from reduced-pyridoxal phosphate to Nile Red in the wild type alpha(2)beta(2) complex by showing decreased reduced pyridoxal phosphate emission with increasing concentrations of Nile Red. Because the concentrations of Nile Red in Fig. 5are not saturating, the decrease in reduced-pyridoxal phosphate fluorescence at saturation must be calculated, as shown in the inset and detailed under ``Materials and Methods.'' Similar analysis was performed with the reduced and holoenzyme forms of the wild type, F280C, and F280S alpha(2)beta(2) complexes. Significant energy transfer was found in all cases, with extrapolated efficiencies of transfer in the range of 0.55-0.85 for wild type and F280C alpha(2)beta(2) complexes. Transfer efficiencies were higher with the holo-alpha(2)beta(2) complexes than with the reduced alpha(2)beta(2) complexes, although the error ranges overlapped. These values for transfer efficiency indicate a separation distance of about 0.75-0.97 times the critical Förster distance, R(o), corresponding to 14-27 Å. This result is consistent with Nile Red binding near Cys-170, which is about 16 Å from pyridoxal phosphate (see Fig. SIB).


Figure 5: Energy transfer from excited reduced pyridoxal phosphate to Nile Red. Reduced pyridoxal phosphate fluorescence emission from 5 µM reduced alpha(2)beta(2) complex was recorded upon direct excitation at 315 nm with 0, 0.4, 0.6, 0.8, and 1.0 µM Nile Red added (top to bottom). The data clearly show the progressive reduction in intensity of pyridoxal fluorescence with increased Nile Red addition. The inset shows the reduction in integrated fluorescence (DeltaF) plotted versus the free Nile Red concentration (calculated as described under ``Materials and Methods''), with the data presented in double-reciprocal format. The reciprocal of the y intercept gives the value of DeltaF at saturation, which yields the value of E, the efficiency of energy transfer at saturation. Here the data indicate that at saturation with Nile Red, the pyridoxal fluorescence would be decreased by 76% or E = 0.76. The experiment was also performed with the holo-alpha(2)beta(2) complex (excitation at 410 nm) and with the holoenzyme and reduced forms of the F280S and F280C alpha(2)beta(2) complex (data not shown).



Nile Red Binding Site: Molecular Modeling

The results above suggest that Nile Red binds in a nonpolar environment in the tunnel near beta subunit residues Cys-170 and Phe-280 and has greater occupancy when the bulky side chain of Phe-280 is replaced by SH or OH. To determine whether a binding site for Nile Red site in this location is compatible with the known three-dimensional structure of the alpha(2)beta(2) complex(15) , we have used molecular modeling (see ``Materials and Methods''). In the current crystallographic model (``closed tunnel model'') of the wild type tryptophan synthase alpha(2)beta(2) complex,^1 the side chains of two adjacent aromatic residues (Tyr-279 and Phe-280) have adopted a configuration that closes the tunnel in this region as shown in Fig. SIB for Phe-280. An alternative ``open tunnel model'' of the alpha(2)beta(2) complex can be obtained by use of molecular modeling to move the Tyr-279 and Phe-280 side chains from the center of the tunnel. A similar, open tunnel conformation has been observed by x-ray crystallography under some experimental conditions.^1 We have used these crystallographic results to model the Nile Red in the open tunnel (Fig. 6). In this position, the center of pyridoxal phosphate is 11 Å from the center of the nearest Nile Red ring and 16.5 Å from the center of the farthest Nile Red ring. Nile Red has some motional freedom in this site and is able to rotate somewhat about the long axis of the molecule. Motion about the short axis (e.g. rotating end to end) is quite restricted. However, it is possible to model Nile Red in this location in the opposite orientation (i.e. with the diethylamino group distal to pyridoxal phosphate). Movement of Nile Red farther down the tunnel toward the alpha site is not possible due to a turn in the tunnel near the alpha/beta interface. However, between this bend and the pyridoxal phosphate, the tunnel has the approximate dimensions 16 Å long, 9 Å wide, and 4.5 Å deep, allowing adequate room for Nile Red which has dimensions 12 Å long, 6 Å wide, and 3.4 Å thick.


Figure 6: Computer graphics modeling of Nile Red in the tryptophan synthase alpha(2)beta(2) complex. The model is based on the x-ray crystallographic results (15) and K. D. Parris, C. C. Hyde, S. A. Ahmed, E. W. Miles, and D. R. Davies, unpublished results. The side chains of Phe-280 and Tyr-279 have been removed to show the open tunnel conformation described under ``Results.'' Close up of the beta site showing the pyridoxal phosphate:Lys-87 Schiff base (PLP), the approximate surface of the tunnel (dots) nearby and Cys-170 (C-170). The Nile Red molecule (NR) (12 Å long, 6 Å wide, and 3.4 Å thick) was generated by computer graphics modeling and positioned in the optimal position in the tunnel (16 Å long, 9 Å wide, and 4.5 Å deep in this region).



The position of Nile Red directly adjacent to Cys-170 is clearly consistent with the Cys-170 and Phe-280 modification and substitution results, as well as with the energy transfer results. This site should be available in the F280C and F280S alpha(2)beta(2) complexes because the large phenylalanine that enters the tunnel in this location has been replaced by a smaller residue. This may be available some of the time in the wild type complex if an equilibrium occurs between open and closed conformations (see ``Discussion'').

Using the closed tunnel model, Nile Red could only be fit in the beta site in a somewhat more restricted site closer to the pyridoxal phosphate in a position that overlaps the site which tryptophan occupies when the pyridoxal phosphate-L-tryptophan Schiff base is present in the beta subunit active site. (^5)In this position, the center of the pyridoxal phosphate ring is 3.5 Å from the nearest Nile Red ring center and about 11 Å from the nitrogen of Nile Red. Nile Red is quite restricted in possible motions in the absence of movements in the protein matrix. Rotation about either the long or short axis of the molecule is very restricted and movement into the tunnel or through the tunnel is not possible. A nonpolar area large enough to contain Nile Red could not be identified either on the surface of the alpha(2)beta(2) complex or at the betabeta interface. One site that could accommodate Nile Red was found in the active site of the alpha subunit near the beginning of the tunnel.

Effects of beta Subunit Active Site Ligands on the Fluorescence Emission of Nile Red

Addition of L-serine, a beta site ligand, results in a modest decrease in the nonpolar emission of Nile Red with the holo-beta subunit (Fig. 7B) and in a much larger decrease (70%) in the nonpolar component with the holo-alpha(2)beta(2) complex (Fig. 7C and Table 3). Although most experiments utilized a very high concentration of L-serine (50 mM), the nonpolar component of Nile Red emission with the holo-alpha(2)beta(2) complex is strongly decreased by much lower concentrations ( Fig. 8and Table 3). Analysis of the L-serine effect by assuming that loss of fluorescence is proportional to L-serine bound yields K(d) = 0.15 mM for L-serine, in good agreement with K(d) = 0.2 mM for L-serine determined by another method(52) . Addition of other beta subunit ligands (D-tryptophan, L-tryptophan, indole, benzimidazole, and benzimidazole + L-serine) to the holo-alpha(2)beta(2) complex also decrease the nonpolar component of Nile Red emission (Table 3). Plots of reciprocal fluorescence versus reciprocal Nile Red concentration in the presence of indole or D-tryptophan indicate noncompetitive inhibition (Fig. 3C).


Figure 7: Effects of ligands on the emission spectra of Nile Red with the alpha subunit (A), the beta subunit (B), and the wild type holo-alpha(2)beta(2) complex (C). Conditions are the same as in Fig. 1. Enzyme alone (curve 1), in the presence of 80 mMDL-alpha-glycerol 3-phosphate (curve 2), in the presence of 50 mML-serine (curve 3), or in the presence of both ligands (curve 4). The order of addition of Nile Red and ligands did not affect the final spectra obtained.




Figure 8: Effect of L-serine concentration on the fluorescence emission of Nile Red with the holo-alpha(2)beta(2) complex. A, fluorescence emission spectra with holo-alpha(2)beta(2) complex (5 µM), Nile Red (1 µM), and L-serine (0-50 mM). B, plot of the change in reciprocal fluorescence emission at 620 nm versus reciprocal concentration of L-serine.



Effects of Allosteric Ligands and of Cleavage on an alpha Subunit Loop on the Fluorescence Emission of Nile Red

alpha-Glycerol 3-phosphate and indole-3-propanol phosphate are analogues of indole-3-glycerol phosphate, the substrate that is cleaved at the active site of the alpha subunit. Addition of alpha-glycerol 3-phosphate dramatically reduces the emission intensity of Nile Red bound to the wild type ( Fig. 7C and Table 3) and F280C and F280S (Table 3) alpha(2)beta(2) complexes but has a very small effects (10% reduction in intensity) on the emission spectra of Nile Red bound to the alpha subunit (Fig. 7A). Indole-3-propanol phosphate has a similar effect on the holo-alpha(2)beta(2) complex (Table 3). Total fluorescence intensity is reduced by one-half, principally due to loss of the nonpolar component. Thus binding of alpha-glycerol 3-phosphate or indole-3-propanol phosphate to the alpha subunit alters the Nile Red binding to the putative beta site. Deconvolution analysis of the spectra reveals that alpha-glycerol 3-phosphate and indole-3-propanol phosphate do not alter the effective polarity of the beta site since the (max) is not significantly altered but the intensity at 626-627 nm is decreased (Table 3).

Tryptic cleavage of the alpha subunit in flexible loop-6 at Arg-188 (51) alters the allosteric properties of the tryptophan synthase alpha(2)beta(2) complex(30, 53) . Fig. 9, A and B, show the fluorescence emission spectra of Nile Red with the nicked alpha subunit and ``nicked'' holo-alpha(2)beta(2) complex, respectively. The emission of Nile Red has a larger nonpolar component with the nicked alpha subunit (Fig. 9A) than with the uncleaved alpha subunit (Fig. 1A and Fig. 7A). In contrast, the fluorescence emission of Nile Red with the nicked alpha(2)beta(2) complex (Fig. 9B and Table 1) is closely similar to that of the alpha(2)beta(2) complex (Fig. 3C and Fig. 7C and Table 3). Spectrum 1 for the nicked alpha(2)beta(2) complex in Fig. 9B is clearly not the sum of spectrum 1 for the beta subunit in Fig. 1B and spectrum 1 for the nicked alpha subunit in Fig. 9A, but instead is very similar to the emission spectra of the holo-alpha(2)beta(2) complex and the holo-beta subunit. This result implies that the site that interacts with Nile Red in the nicked alpha subunit is not available in the nicked alpha(2)beta(2) complex.


Figure 9: Effects of ligands on the emission spectra of the nicked alpha subunit (A) and of the nicked alpha(2)beta(2) complex (B). Conditions are the same as in Fig. 1. Nicked alpha subunit or nicked alpha(2)beta(2) complex alone (curve 1), in the presence of 80 mMDL-alpha-glycerol 3-phosphate (curve 2), in the presence of 50 mML-serine (curve 3), or in the presence of both ligands (curve 4).



alpha-Glycerol 3-phosphate has negligible effects on the Nile Red fluorescence with either alpha subunit (Fig. 7A) or nicked alpha subunit (Fig. 9A). alpha-Glycerol 3-phosphate has a much smaller effect on the Nile Red fluorescence with the nicked holo-alpha(2)beta(2) complex (Fig. 9B and Table 3) than with the holo-alpha(2)beta(2) complex (Fig. 7C and Table 3). Addition of L-serine greatly alters the Nile Red fluorescence of the holo-alpha(2)beta(2) complex (Fig. 7C) and the nicked holo-alpha(2)beta(2) complexes and (Fig. 9B). Thus Nile Red fluorescence with the nicked holo-alpha(2)beta(2) complex appears to have a normal response to a beta subunit ligand but is desensitized to the effects of an allosteric alpha subunit ligand.


DISCUSSION

We will first discuss evidence for the location of Nile Red in the tryptophan synthase tunnel in the beta site and then discuss how this information can be used to understand the effects of active site (beta subunit) and allosteric (alpha subunit) ligands and of cleavage of the alpha subunit loop-6 on the geometry of the tunnel.

Nile Red Binding Site

The fluorescence emission spectrum of Nile Red with the holo-alpha(2)beta(2) complex (Fig. 1C and Table 1) clearly indicates that the fluorescence is due to more than one component. One component has properties essentially the same as Nile Red in aqueous solution, with emission maximum at 665 nm. The second component represents protein bound dye and is well described by emission from a single class of sites, with polarity properties similar to isopropanol(18) . The similarity of the wavelength maxima and intensities of the nonpolar component of Nile Red emission with the holo-beta subunit and holo-alpha(2)beta(2) complex indicates that Nile Red binds primarily to a beta subunit site in the alpha(2)beta(2) complex.

To determine the properties and location of the Nile Red binding site, a number of experimental approaches were used. Titration of tryptophan synthase with Nile Red to determine binding strength and stoichiometry is not possible in the usual manner, because Nile Red is much less soluble in aqueous solution than in nonpolar solvents. This is reflected in a partition coefficient of about 200 between nonpolar organic solvents and water(17) . By contrast, other fluorophores that have polarity-sensitive emission, such as 1-anilinonaphthalene-8-sulfonic acid, are quite water-soluble due to an ionized group, but only partition significantly into nonpolar solvents in the presence of a counterion, such as an amine(54) . Nile Red may have advantages over other probes that contain charged groups for probing accessible, but nonpolar sites in proteins. The spectral properties of Nile Red are advantageous for the study of pyridoxal phosphate-containing enzymes such as tryptophan synthase. The absorbance and emission of Nile Red, unlike those of most fluorescent probes, occur in a wavelength range that is substantially removed from the broad absorbance and emission peaks of the enzyme. The drawback imposed by the nonpolar nature of the probe is that the range of aqueous concentrations available for an assay is limited.

The location of the bound Nile Red within the beta subunit was investigated using forms of the alpha(2)beta(2) complex altered specifically by chemical modification and site-directed mutagenesis. The nonpolar component of Nile Red emission is significantly reduced by the presence of a bulky group at position 170 of the beta subunit (see C170-NEM, C170F, C170W on Fig. 2A and Table 1). Replacing the bulky side chain of Phe-280, which enters the tunnel in this region, greatly increases the nonpolar component of Nile Red emission (see F280C and F280S in Fig. 2B and Table 1). These results provide additional strong support for a location for Nile Red in this region of the tunnel near Cys-170 and Phe-280.

The iodide quenching results ( Fig. 4and Table 2) support a location for the Nile Red binding site in the holo-beta subunit, since the Stern-Volmer quenching constant (K) obtained for the holo-alpha(2)beta(2) complex is quite similar to that obtained for the holo-beta subunit and quite different from that obtained for the alpha subunit. The site of interaction of Nile Red with the alpha(2)beta(2) complex is clearly different from the alpha subunit site, because the nonpolar component of Nile Red emission with the free alpha subunit has a lower emission maximum (619 nm, Table 1) and is completely shielded from iodide quenching (Table 2), indicating a nonpolar site buried in the alpha subunit. The possibility that this site is the active site of the alpha subunit is consistent with earlier studies which demonstrated that acrylamide does not quench the fluorescence of indole-3-propanol phosphate bound to the alpha subunit(40) . This site appears to be unavailable to Nile Red in the native or nicked holo-alpha(2)beta(2) complex.

The quenching observed with the holo-alpha(2)beta(2) complex appears largely due to a static rather than a collisional mechanism. While this precluded use of the quenching data to determine accessibility of the bound Nile Red quantitatively,^4 the results do indicate that the Nile Red is removed from free contact with soluble KI since KI can collisionally quench Nile Red emission if it can contact Nile Red. A site removed from open aqueous solvent is consistent with the 625-nm emission maximum found for this component (Table 1), which indicates a nonpolar environment. Furthermore, alteration of the side chain of beta subunit residue 280 has similar effects on KI quenching of Nile Red and pyridoxal phosphate, suggesting that the two fluorophores are close to each other and to residue 280. The combined results provide evidence that Nile Red binds in the holo-beta subunit in a nonpolar site, such as the milieu of the tunnel that leads from the active site of the alpha subunit to the active site of the beta subunit(15) .

Location of Nile Red in this portion of the tunnel should allow for nonradiative energy transfer from pyridoxal phosphate to Nile Red, since the spectral overlap is satisfactory. Analysis of energy transfer efficiency (illustrated in Fig. 5) yielded an estimated separation distance range of 14-27 Å for all of the holoenzyme and reduced forms of the wild type, F280C and F280S alpha(2)beta(2) complexes, assuming no major orientational effects. This result is clearly consistent with a Nile Red binding site near the Cys-170 residue in this portion of the tunnel.

Molecular modeling of Nile Red binding located a possible binding site of adequate volume in the beta subunit near pyridoxal phosphate in the open tunnel model (see ``Results'' and Fig. 6). This site is nonpolar and removed from contact with bulk solvent and thus consistent with the spectral properties of the Nile Red emission ( Fig. 1and Table 1), the iodide quenching data ( Fig. 4and Table 2), and the reduction in fluorescence observed with active site ligands (Fig. 7C and 8 and Table 3). The orientation of Nile Red in this site is adjacent to Cys-170 and is consistent with the results of cysteine modification and mutational replacement of Cys-170 shown in Fig. 2A. Because this site is blocked by intrusion of Phe-280 into the tunnel (see Fig. SI), this site should be more available with a smaller side chain on residue 280, which is consistent with the increased fluorescence emission of Nile Red observed upon replacement of Phe-280 with smaller residues (Fig. 2B). In addition this site dictates a pyridoxal phosphate-Nile Red separation distance that is consistent with the energy transfer results. Thus, we suggest that the location of Nile Red in Fig. 6is the most likely binding site for Nile Red in the alpha(2)beta(2) complex. The holo-alpha(2)beta(2) complex in solution probably contains an equilibrium distribution of open tunnel and closed tunnel forms. We suggest that Nile Red binds preferentially to the open tunnel form. Molecular modeling located a second, more restricted site closer to pyridoxal phosphate in the closed tunnel structure and a site in the alpha subunit, essentially at the beginning of the tunnel (not shown). The site located in the alpha subunit may be the origin of the nonpolar component of Nile Red interaction with the free alpha subunit, but appears to be unavailable in the alpha(2)beta(2) complex.

Allosteric Ligands Alter Nile Red Fluorescence in the Holo-alpha(2)beta(2) Complex but Not in the Nicked Holo-alpha(2)beta(2) Complex

Although there is much indirect evidence that allosteric interactions in tryptophan synthase result from ligand-induced conformational changes that are transmitted from one subunit to the other(20, 55, 56, 57) , there is little direct physical evidence for where the conformational change occurs. Our finding that alpha site ligands (alpha-glycerol 3-phosphate and indole-3-propanol phosphate) dramatically reduce the nonpolar component of Nile Red emission with the holo-alpha(2)beta(2) complex (Fig. 7A and Table 3) provides direct evidence that the allosteric ligands alter the conformation of the Nile Red binding site near the active site of the beta subunit (Fig. 6). The change in Nile Red fluorescence could be due to either reduced occupancy by Nile Red or to decreased quantum yield of Nile Red as the consequence of quenching from nearby protein groups. Either reduced occupancy or quenching could result from a constriction of the Nile Red binding site in the beta subunit induced by alpha site ligands. This constriction could result from conversion of the alpha(2)beta(2) complex into the closed tunnel form described above by intrusion of Phe-280 and Tyr-279 into the tunnel (see Fig. SIB). Phe-280 and Tyr-279 might thus act as an allosteric gate, coupled to enzymatic reaction steps(2) .

We have previously reported that cleavage of the alpha subunit at Arg-188 in a flexible loop-6 prevents the transmission of ligand-induced conformational changes from the alpha subunit to the beta subunit(30, 53) . Our new finding that this tryptic cleavage desensitizes the holo-alpha(2)beta(2) complex to the allosteric effects of alpha-glycerol 3-phosphate on the nonpolar component of Nile Red emission (Fig. 9B and Table 3) provides direct evidence that cleavage of loop-6 prevents ligand-induced communication between the alpha subunit and a specific site in the beta subunit via the tunnel. It is noteworthy that the nonpolar component of Nile Red emission is enhanced in the nicked alpha subunit relative to that in the uncleaved alpha subunit (Fig. 9A versus 7A) but is not altered in the nicked alpha(2)beta(2) complex (Fig. 9BversusFig. 7C). These results indicate that proteolytic cleavage of the separate alpha subunit alters the conformation and makes the nonpolar interior more accessible to Nile Red, whereas the same cleavage of the complex does not alter the accessibility of Nile Red in the nicked alpha(2)beta(2) complex. These results imply either that the conformation of the nicked alpha subunit is stabilized by interaction with the beta subunit or that the nonpolar interior of the nicked alpha subunit is less accessible to Nile Red in the nicked alpha(2)beta(2) complex or both.

Effects of beta-Site Ligands on Nile Red Fluorescence

Addition of L-serine to the holo-alpha(2)beta(2) complex (Fig. 7C and Fig. 8and Table 3) and to the nicked holo-alpha(2)beta(2) complex (Fig. 9B and Table 3) results in a dramatic decrease in total Nile Red fluorescence intensity, principally due to loss of emission from the nonpolar component. Thus formation of an enzyme-substrate complex at the beta site either makes the nonpolar interior of the alpha(2)beta(2) complex less accessible to Nile Red or alters the environment of the Nile Red. This result is consistent with previous suggestions that L-serine induces a conformational change in the beta subunit in the alpha(2)beta(2) complex or stabilizes an active conformation. These suggestions were based on the ability of L-serine to stabilize the alpha(2)beta(2) complex from dissociation (58, 59) and to trigger the activation of the alpha site in the alpha(2)beta(2) complex(11, 14, 57, 60) . This activation of the alpha subunit has been attributed to a conformational change in the beta subunit that is induced by conversion of the Schiff base of L-serine to the Schiff base of aminoacrylate. Our results imply that this conformational change alters the geometry of the putative Nile Red site in the tunnel near pyridoxal phosphate (Fig. 6). The reaction of L-serine with the holo-beta subunit (Fig. 7B) results in a smaller decrease in fluorescence intensity of Nile Red than the reaction with the holo-alpha(2)beta(2) complex (Fig. 7C). The reaction of L-serine with the beta subunit leads to the accumulation of the Schiff base of L-serine, not to the Schiff base of aminoacrylate(61) . Our results imply that formation of the Schiff base of aminoacrylate results in a larger restriction of the tunnel than does formation of the Schiff base of L-serine.

Other beta site ligands (D- and L-tryptophan, indole, benzimidazole, and L-serine + benzimidazole) also markedly decrease the fluorescence intensity of Nile Red with the holo-alpha(2)beta(2) complex (Table 3). Because these ligands bind at or near the putative Nile Red binding site shown in Fig. 6, these beta site ligands might be expected to be competitive inhibitors of Nile Red binding. However, fluorescence titration results indicate that indole and L-tryptophan are noncompetitive inhibitors of Nile Red (Fig. 3C). A probable explanation for these results is that Nile Red binds reversibly to the open tunnel form of the beta site and is released upon addition of beta site ligands that induce the closed tunnel conformation of the beta subunit that does not bind Nile Red. Thus Nile Red and beta site ligands do not compete because they do not bind to the same form of the enzyme. Similarly, the substrates used in the assay of activity in the alphabeta reaction (indole-3-glycerol phosphate and L-serine) induce the closed tunnel conformation of the beta subunit that does not bind Nile Red. Thus Nile Red cannot bind and cannot inhibit the enzyme under steady-state reaction conditions. Rapid kinetic experiments under single turnover conditions using enzyme preincubated with Nile Red might detect effects of Nile Red in the tunnel on the initial rate.

Conclusions

Nile Red binds to a nonpolar site in the tryptophan synthase beta subunit and alpha(2)beta(2) complex. Based on our model for a location of Nile Red in the tunnel near the active site of the beta subunit (Fig. 6) and our finding that allosteric alpha subunit ligands decrease the fluorescence of Nile Red ( Table 3and Fig. 7), we can speculate that allosteric ligands induce conformational changes that result in partial closure of the tunnel in this region. This proposal is consistent with the observed structure tightening effect of alpha-glycerol 3-phosphate(62) . Our finding that beta subunit ligands decrease the fluorescence of Nile Red suggests that these ligands also induce tunnel closure in this region and provide a possible structural basis for the postulated ligand-induced conversion of the beta subunit in the alpha(2)beta(2) complex from an ``open'' to a ``closed'' form(14, 62, 63) . The results with the nicked alpha(2)beta(2) complex allow us to differentiate the effects of allosteric ligands from those of active site ligands. That is, proteolysis reduces the effects of allosteric ligands but not of active site ligands. Thus active site (beta subunit) and allosteric (alpha subunit) ligands alter the geometry of the tunnel near the Nile Red site by independent mechanisms that can be distinguished by studies with the nicked alpha(2)beta(2) complex. Our results support the suggestion based on the crystal structure that Phe-280 may act as an allosteric gate, coupled to enzymatic reaction steps(2) , and demonstrate that Nile Red binding near Phe-280 serves as a sensitive reporter group of specific effects of ligands on this region of the tunnel.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: National Institutes of Health, Bldg. 8, Rm. 225, Bethesda, MD 20892-0830. Tel.: 301-496-4033 (D. L. S.) or 301-496-2763 (E. W. M.); Fax: 301-402-0240.

(^1)
C. C. Hyde, K. D. Parris, S. A. Ahmed, E. W. Miles, and D. R. Davies, unpublished results.

(^2)
The abbreviations used are: NEM, N-ethylmaleimide; MMTS, methyl methanethiolsulfonate; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.

(^3)
E. coli CB 149/pSTB7, which was originally developed by Dr. Ronald Bauerle, has been deposited at the American Type Culture Collection for distribution as ATCC 38745.

(^4)
The solvent accessibility of fluorophores is often estimated by comparing the bimolecular rate constant for quenching (k(q)) of a bound fluorophore with that obtained in free solution. The value of k(q) is determined from the Stern-Volmer constant (K) and the fluorescence lifetime ((0)) by the equation: K = k(q) times (0). The value of k(q) for Nile Red in dimethylformamide was 4.4 times 10^9M s (K = 19.5 M and (0) = 4.4 ns), consistent with diffusion-limited, efficient quenching of Nile Red by KI. The comparable value of k(q) for Nile Red in the holo-alpha(2)beta(2) complex was 0.33 times 10^9M s (K= 1.5 M, (0) = 4.5 ns). This represents a 10-fold reduction in apparent bimolecular rate constant compared with Nile Red free in solution. This value of k(q) is similar to the value of k(q) previously reported for KI quenching of pyridoxal phosphate in the holo-alpha(2)beta(2) complex, 0.52 times 10^9M s(40) . The similarity of the values of k(q) for Nile Red and pyridoxal phosphate might indicate that these two fluorophores have similar exposures in the alpha(2)beta(2) complex. A valid, quantitative estimate of solvent exposure of a fluorophore can only be obtained if the quenching mechanism is collisional. This is not the case with the Nile Red-holo-alpha(2)beta(2) complex, since a 30% quench of fluorescence intensity was associated with a 3.5% decrease in lifetime (data not shown). Thus k(q) values calculated using (o) and K are not valid bimolecular rate constants and cannot be used to compare exposure of two sites. It may be observed that many published studies of binding site exposure rely on (o) and a steady-state K without demonstrating that quenching is collisional (e.g.(40) ).

(^5)
K. D. Parris, C. C. Hyde, S. A. Ahmed, E. W. Miles, and D. R. Davies, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. C. Craig Hyde for his suggestions for mutagenesis and his generosity in preparing Fig. SIB, Dr. Jay Knutson for the time resolved fluorescence measurements and many helpful discussions, and Peter McPhie and Jan Wolff for advice and comments.


REFERENCES

  1. Ovadi, J. (1991) J. Theor. Biol. 152, 1-22 [Medline] [Order article via Infotrieve]
  2. Stroud, R. M. (1994) Nature Struct. Biol. 1, 131-134 [Medline] [Order article via Infotrieve]
  3. Miles, E. W., Ahmed, S. A., Hyde, C. C., Kayastha, A. M., Yang, X.-J., Ruvinov, S. B., and Lu, Z. (1994) in Molecular Aspects of Enzyme Catalysis (Fukui, T., and Soda, K., eds) pp. 127-146, Kodansha, Ltd., Tokyo
  4. Miles, E. W. (1991) Adv. Enzymol. Relat. Areas Mol. Biol. 64, 93-172 [Medline] [Order article via Infotrieve]
  5. Miles, E. W. (1979) Adv. Enzymol. 49, 127-186 [Medline] [Order article via Infotrieve]
  6. Swift, S., and Stewart, G. S. (1991) Biotechnol. Genet. Eng. Rev. 9, 229-294 [Medline] [Order article via Infotrieve]
  7. Yanofsky, C., and Crawford, I. P. (1972) in The Enzymes (Boyer, P. D., ed) Vol. 7, 3rd Ed., pp. 1-31, Academic Press, New York _
  8. Yanofsky, C., and Rachmeler, M. (1958) Biochim. Biophys. Acta 28, 641-642
  9. DeMoss, J. A. (1962) Biochim. Biophys. Acta 62, 279-293 [CrossRef][Medline] [Order article via Infotrieve]
  10. Matchett, W. H. (1974) J. Biol. Chem. 249, 4041-4049 [Abstract/Free Full Text]
  11. Anderson, K. S., Miles, E. W., and Johnson, K. A. (1991) J. Biol. Chem. 266, 8020-8033 [Abstract/Free Full Text]
  12. Dunn, M. F., Aguilar, V., Brzovic', P. S., Drewe, W. F. J., Houben, K. F., Leja, C. A., and Roy, M. (1990) Biochemistry 29, 8598-8607 [Medline] [Order article via Infotrieve]
  13. Lane, A. N., and Kirschner, K. (1991) Biochemistry 30, 479-484 [Medline] [Order article via Infotrieve]
  14. Brzovic', P. S., Ngo, K., and Dunn, M. F. (1992) Biochemistry 31, 3831-3839 [Medline] [Order article via Infotrieve]
  15. Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871 [Abstract/Free Full Text]
  16. Schlicting, I., Yang, X.-J., Miles, E. W., Kim, A. Y., and Anderson, K. A. (1994) J. Biol. Chem. 269, 26591-26593 [Abstract/Free Full Text]
  17. Greenspan, P., Mayer, E. P., and Fowler, S. D. (1985) J. Cell Biol. 100, 965-973 [Abstract]
  18. Sackett, D. L., and Wolff, J. (1987) Anal. Biochem. 167, 228-234 [Medline] [Order article via Infotrieve]
  19. Sackett, D. L., Knutson, J. R., and Wolff, J. (1990) J. Biol. Chem. 265, 14899-14906 [Abstract/Free Full Text]
  20. Kawasaki, H., Bauerle, R., Zon, G., Ahmed, S. A., and Miles, E. W. (1987) J. Biol. Chem. 262, 10678-10683 [Abstract/Free Full Text]
  21. Kirschner, K., Wiskocil, R. L., Foehn, M., and Rezeau, L. (1975) Eur. J. Biochem. 60, 513-523 [Abstract]
  22. Haberland, M. E., and Reynolds, J. A. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 2313-2316 [Abstract]
  23. Yang, X.-J., Ruvinov, S. B., and Miles, E. M. (1992) Protein Exp. Purif. 3, 347-354 [Medline] [Order article via Infotrieve]
  24. Miles, E. W., Kawasaki, H., Ahmed, S. A., Morita, H., Morita, H., and Nagata, S. (1989) J. Biol. Chem. 264, 6280-6287 [Abstract/Free Full Text]
  25. Yang, X.-J., and Miles, E. W. (1993) J. Biol. Chem. 268, 22269-22272 [Abstract/Free Full Text]
  26. Fluri, R., Jackson, L. E., Lee, W. E., and Crawford, I. P. (1971) J. Biol. Chem. 246, 6620-6624 [Abstract/Free Full Text]
  27. Miles, E. W., and Moriguchi, M. (1977) J. Biol. Chem. 252, 6594-6599 [Medline] [Order article via Infotrieve]
  28. Miles, E. W., Houck, D. R., and Floss, H. G. (1982) J. Biol. Chem. 257, 14203-14210 [Abstract/Free Full Text]
  29. Miles, E. W., and Higgins, W. (1978) J. Biol. Chem. 253, 6266-6269 [Abstract]
  30. Miles, E. W. (1991) J. Biol. Chem. 266, 10715-10718 [Abstract/Free Full Text]
  31. Miles, E. W., Bauerle, R., and Ahmed, S. A. (1987) Methods Enzymol. 142, 398-414 [Medline] [Order article via Infotrieve]
  32. Creighton, T. E. (1970) Eur. J. Biochem. 13, 1-10 [Medline] [Order article via Infotrieve]
  33. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Second Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  34. Chung, C. T., Niemela, S. L., and Miller, R. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2172-2175 [Abstract]
  35. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492 [Abstract]
  36. Russel, M., Kidd, S., and Kelley, M. R. (1986) Gene (Amst.) 45, 333-338 [CrossRef][Medline] [Order article via Infotrieve]
  37. Knutson, J. R. (1988) in Proceedings of Society of Photo-optical Instrumentation Engineers (Lakowitz, J., ed) Vol. 909, pp. 51-60, Bellingham, WA
  38. Knutson, J. R., Wallbridge, D. G., and Brand, L. (1982) Biochemistry 21, 4671-4679 [Medline] [Order article via Infotrieve]
  39. Jansson, P. A. (1984) Deconvolution with Applications in Spectroscopy (Jansson, P. A., ed) Academic Press, New York
  40. Lane, A. N. (1983) Eur. J. Biochem. 133, 531-538 [Abstract]
  41. Cheung, H. C. (1991) in Topics in Fluorescence Spectroscopy (Lakowicz, J. R., ed) Vol. 2, pp. 127-176, Plenum Press, New York _
  42. Benesi, H. A., and Hildebrand, J. H. (1949) J. Am. Chem. Soc. 71, 2703-2707
  43. Connors, K. A. (1987) Binding Constants , John Wiley & Sons, New York
  44. Connolly, M. L. (1983) J. Appl. Crystallogr. 16, 548-558 [CrossRef]
  45. Jones, T. A. (1978) J. Appl. Crystallogr. 11, 268-172 [CrossRef]
  46. Miles, E. W. (1970) J. Biol. Chem. 245, 6016-6025 [Abstract/Free Full Text]
  47. Miles, E. W., and Higgins, W. (1980) Biochem. Biophys. Res. Commun. 93, 1152-1159 [Medline] [Order article via Infotrieve]
  48. Smith, D. J., Maggio, E. T., and Kenyon, G. L. (1975) Biochemistry 14, 766-771 [Medline] [Order article via Infotrieve]
  49. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70 [Medline] [Order article via Infotrieve]
  50. Lahti, R., Pohjanoksa, K., Pitkaranta, T., Heikinheimo, P., Salminen, T., Meyer, P., and Heinonen, J. (1990) Biochemistry 29, 5761-5766 [Medline] [Order article via Infotrieve]
  51. Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835 [Medline] [Order article via Infotrieve]
  52. Yang, X.-J., and Miles, E. W. (1992) J. Biol. Chem. 267, 7520-7528 [Abstract/Free Full Text]
  53. Ruvinov, S. B., and Miles, E. W. (1992) FEBS Lett. 299, 197-200 [CrossRef][Medline] [Order article via Infotrieve]
  54. Flanagan, M. T., and Ainsworth, S. (1968) Biochim. Biophys. Acta 168, 16-26 [Medline] [Order article via Infotrieve]
  55. Houben, K. F., and Dunn, M. F. (1990) Biochemistry 29, 2421-2429 [Medline] [Order article via Infotrieve]
  56. Kirschner, K., Weischet, W., and Wiskocil, R. L. (1975) in Protein-Ligand Interaction (Sund, H., and Blaver, G., eds) Walter de Gruyter & Co., Berlin
  57. Kirschner, K., Lane, A. N., and Strasser, A. W. M. (1991) Biochemistry 30, 472-478 [Medline] [Order article via Infotrieve]
  58. Creighton, T. E., and Yanofsky, C. (1966) J. Biol. Chem. 241, 980-990 [Abstract/Free Full Text]
  59. Lane, A. N., Paul, C. H., and Kirschner, K. (1984) EMBO J. 3, 279-287 [Abstract]
  60. Dunn, M. F., Brzovic', P. S., Leja, C., Houben, K., Roy, M., Aguilar, A., and Drewe, W. F., Jr. (1991) in Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors (Fukui, T., Kagamiyama, H., Soda, K., and Wada, H., eds) pp. 257-264, Pergamon Press, New York
  61. Drewe, W. J., and Dunn, M. F. (1985) Biochemistry 24, 3977-3987 [Medline] [Order article via Infotrieve]
  62. Strambini, G. B., Cioni, P., Peracchi, A., and Mozzarelli, A. (1992) Biochemistry 31, 7535-7542 [Medline] [Order article via Infotrieve]
  63. Ahmed, S. A., Ruvinov, S. B., Kayastha, A. M., and Miles, E. W. (1991) J. Biol. Chem. 266, 21540-21557

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