©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fluorescence Resolution of the Intrinsic Tryptophan Residues of Bovine Protein Tyrosyl Phosphatase (*)

(Received for publication, August 10, 1994; and in revised form, December 16, 1994)

Christine Pokalsky (1) Peter Wick (1) Etti Harms (2) Fred E. Lytle (1)(§) Robert L. Van Etten (1)(§)

From the  (1)Departments of Chemistry and (2)Biological Sciences, Purdue University, West Lafayette, Indiana 47907

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Fluorescence steady-state and lifetime measurements have been performed that permit the differentiation of the 2 intrinsic tryptophan residues in bovine low molecular weight phosphotyrosyl protein phosphatase (BPTP). Spectral information was obtained by use of two single-tryptophan mutant proteins, W39F and W49F, and the double mutant protein W39,49F. Fluorescence measurements show that Trp is characterized by a large blue shift, a low quantum yield, and a shorter mean lifetime compared to Trp. Solute fluorescence quenching studies of W39F reveal that Trp is highly exposed to the aqueous environment. In contrast, Trp is situated within a hydrophobic core and is only partially accessible to quenching agents such as acrylamide, iodide ion, and cesium ion. The fluorescence contributions of Trp and Trp are additive, and their sum is equivalent to that observed for wild type BPTP. Calculated intramolecular distances between Trp or Trp and a 5-[[(acetylamino)-ethyl]amino]naphthalene-1-sulfonate group covalently bound at Cys or Cys of the respective protein mutants, place Trp within 10 Å and Trp at least 20 Å from the active site. The fluorescence decay of the single tryptophan mutants and, surprisingly, wild type BPTP were each adequately fitted as biexponentials. The latter is a consequence of the imprecision involved in determining actual minima in a three- and four-exponential fitting. Comparison of quenching results of wild type BPTP with those of the single tryptophan mutant proteins indicates that minor fluorescence components, easily resolved using a biexponential fitting for the mutant proteins, are unresolvable for wild type BPTP. These minor components skewed the weighted magnitudes and induced perturbations in lifetimes for the tryptophan fluorescence of wild type BPTP, which directly influenced the calculated values of K and k.


INTRODUCTION

Protein tyrosine phosphorylation and dephosphorylation are cellular mechanisms that regulate cell growth, proliferation, and transformation (Hunter, 1987; Lau et al., 1989; Fischer et al., 1991; Charbonneau and Tonks, 1992). The phosphorylated state of a protein is kept in balance by phosphotyrosyl kinases and phosphatases. There are several classes of protein tyrosine phosphatases (PTPases) (^1)including high molecular weight, membrane-associated and receptor-linked PTPases, and distinct low molecular weight cytoplasmic PTPases that may mediate the above events intracellularly, thus influencing signal transduction and other diverse cellular processes. Low molecular weight PTPases dephosphorylate phosphotyrosyl peptides and proteins including angiotension, tyrosine kinase P40, erythrocyte band 3, and epidermal growth factor receptor (Chernoff and Li, 1985; Boivin and Galand, 1986; Waheed et al., 1988; Ramponi et al., 1989; Zhang and Van Etten, 1990), but exhibit little or no activity toward phosphoseryl or phosphothreonyl peptides. Bovine phosphotyrosyl protein phosphatase (BPTP) is a monomeric 18-kDa protein containing a single catalytic domain and may be considered to be a prototypical low molecular weight PTPase.

The catalytic mechanism by which BPTP and other cytoplasmic PTPases proceed involves the formation of a covalent phosphoenzyme intermediate that is attacked by water during the rate-limiting step (Saini et al., 1981; Zhang and Van Etten, 1991). As a result of extensive studies of non-fusion, recombinant BPTP, it is known that both Cys and Cys are located at the active site and that Cys acts as the nucleophile that attacks the phosphate function of phosphorylated substrates (Davis et al., 1994). Such crucial knowledge of the active site residues and the mechanism by which BPTP acts has been intensely pursued. However, the role of protein folding processes leading to the native tertiary structure has been largely neglected. The identification of suitable reporter groups is critically important for such investigations.

Crystallography provides a plethora of knowledge about the tertiary structure of proteins but seldom permits the determination of local structural fluctuations during folding or catalysis. Tryptophan residues can often be used as fluorescent probes to investigate the dynamic nature of proteins. Moreover, methods for studying rapid folding and denaturation reactions of proteins such as BPTP require sensitive analytical signals that respond rapidly but do not alter the behavior of the protein. Techniques that involve mapping of exposed and buried residues (Kronmann and Robbins, 1970) are often used to elucidate protein structure and to study conformational perturbations. Tryptophan residues, which are environmentally sensitive and constitute only 1.1 mol % of typical proteins, are valuable probes of protein structure. While many proteins contain only 1 tryptophan residue, it is not uncommon to observe proteins that include multiple tryptophan residues within their amino acid sequence. In addition, the purposeful introduction of a tryptophan into a specific site of a protein can provide an extremely valuable probe of structure and function (Stole and Bryant, 1994).

Wild type (WT) BPTP contains 2 tryptophan residues, at positions 39 and 49 (Wo et al., 1992). We were interested in the possibility that these residues could be used as probes of structure and function. To this end, it was necessary to characterize the fluorescence properties of the intrinsic tryptophan residues of the enzyme. With the advent of site-directed mutagenesis, it is now often possible to achieve unambiguous assignments of spectral signals due to the tryptophan residues of proteins, a process that was once hindered by the existence of multiple tryptophan fluorescence signals and lifetimes (Waldman et al., 1987; Royer et al., 1990; Mas et al., 1994). Here we describe the use of tryptophan mutant proteins and quenching studies to determine the fluorescence characteristics of the tryptophan residues of BPTP, thus deriving information about their immediate microenvironments and providing a basis for their utilization as reporter groups in studying the folding and catalytic activity of BPTP.


EXPERIMENTAL PROCEDURES

Materials

Ultrapure grades of acrylamide (Life Technologies, Inc.), CsCl (Life Technologies, Inc.), and Tris(hydroxymethyl)aminomethane (THAM grade, Fisher Scientific) were used without further purification. KCl (Mallinckrodt), KI (Mallinckrodt), Na(2)S(2)O(3)bullet5 H(2)O (Fisher Scientific), and bovine serum albumin (Sigma) were all of high purity and used as obtained. Indole, 1- and 5-methylindole were purchased from Aldrich and used without further purification. 3-Phosphoglycerate kinase, from bakers' yeast (Sigma) was received as a lyophilized powder containing citrate buffer salts. The buffer for 3-phosphoglycerate kinase was changed by dialysis to 0.01 M Tris at pH 7, 0.09 M NaCl, and 1 mM EDTA and loaded onto a Sephadex G-25 size exclusion column (1.5 times 4 cm) pre-equilibrated with the above mentioned buffer. 5-[2-(2-Iodoacetamido)ethylamino]-1-naphthalenesulfonic acid (IAEDANS) was purchased from Molecular Probes.

Instrumentation

Steady-state fluorescence was detected using a Photon Technology Inc.(Brunswick, NJ) spectrofluorometer model LS-100 (PTI). Excitation was at 295 nm with a bandwidth of 2 nm, and emission was monitored from 300 to 400 nm (bandwidth of 5 nm), unless otherwise noted. Samples were contained in a NSGT-52 dual pathlength (1 cm times 4 mm) cell which was positioned within the apparatus such that the excitation light entered along the 1-cm pathlength, and the short path portion was used to reduce any inner filter effects. Lifetime determinations were performed with a time-correlated single-photon counting instrument that consisted of a synchronously pumped, cavity dumped, frequency doubled (Quantum Technology LiIO(3) crystal) dye laser (Spectra Physics model 375B dye laser and model 344 cavity dumper). The dye laser was operated with rhodamine 590 (Kodak) driven by a mode-locked Nd:YAG laser (Spectra Physics series 3000). This arrangement yields 10-20 ps wide pulses at 4 MHz with a tunable wavelength from 285 to 320 nm. Typically experiments were run with the wavelength set at 295 nm. The detector for the system was a Burle 8850 photomultiplier biased at a potential of -2250 V and operated in the single photon counting mode. The output of the photomultiplier tube was connected to a constant fraction discriminator (Tennelec 455). A fast photodiode (Texas Instruments TIED 56) biased with a potential of -125 V and wired for a 200 ps risetime (Lytle et al., 1980) was employed to provide a trigger coincident with sample excitation. Spectral resolution was established by a Jarrell Ash 1/4 meter grating monochromator with a bandwidth of approximately 10 nm. The time correlation between excitation and emission events was provided by a Tennelec model 864 Time-to-Amplitude Converter. The time-to-amplitude converter output pulse was processed by a multichannel analyzer (Tennelec PCA-Multiport-E) operating in the pulse height analysis mode. Timing hardware gave resolutions of 24-96 ps/channel. The impulse response of the instrument was 1.2 ns. Darkcounts and scatter were the primary contributors to the blank. The excitation beam was vertically polarized, and sample emission was observed through a Glan-Thomson polarizer at 55° to remove polarization effects. Control of the data acquisition system was provided by an IBM PS/2 model 90, and data were transferred to the computer for further processing.

Expression and Preparation of WT BPTP and BPTP Mutant Proteins

Site-specific mutations within the BPTP gene were generated by the method of Vandeyar et al.(1988). A 765-base pair XbaI-BamHI fragment containing the BPTP gene was transferred from plasmid pVEBH4 (Wo et al., 1992) into the corresponding sites of bacteriophage M13mp18. The single-stranded DNA of the resulting M13mp18 derivative was the template for the mutagenesis. The following primers were used to initiate second strand synthesis: 5`-CAGATAATTTCGTCATTGAC-3`, 5`-GCTGTTTCTGACTTCAACGTGGGC-3`, and 5`-CAGATAATTTCGTCATTGACAGTGGCGCTGTTTCTGACTTCAACGTGGGC3`. In each case, the codon changes were TGG to TTC to convert Trp and/or Trp to Phe, thus obtaining the single mutants W39F and W49F, and the double mutant W39,49F. Mutagenesis was performed using the T7 in vitro mutagenesis kit from United States Biochemicals Corp. Initial screening for the presence of the desired mutation was performed using single base nucleotide sequence reactions (in this case, ``T-tracking''). The mutant BPTP XbaI-BamHI fragments were excised from the bacteriophage M13mp18 derivatives and inserted into the analogous sites of the expression vector pET11d and subsequently transformed into the Escherichia coli B strain BL21(DE3) for overexpression of the mutant bovine phosphotyrosyl protein phosphatase. The complete nucleotide sequences of the mutant BPTP genes in the final plasmid constructs were determined in order to verify the mutation as well as the integrity of the remainder of the gene.

Wild type BPTP and mutant proteins were expressed and purified using the methods previously described (Davis et al., 1994). WT BPTP and the mutant proteins W39F, W49F, and W39,49F were subjected to denaturation using 8 M guanidine hydrochloride in 50 mM 3,3-dimethylglutarate, pH 7.0, at 4 °C for 2 h. Then 30 mM dithiothreitol was added and the solution stirred for 4 h. The enzyme was renatured by passage through a Sephadex G-25 column pre-equilibrated at 4 °C using 100 mM NaOAc buffer, 0.08 M NaCl, pH 5.0, and fractions of 0.5 ml were collected and assayed for activity. Fractions containing activity were pooled and loaded on to a 12.5 times 0.9-cm SP-Sephadex C-50 column pre-equilibrated with pH 5 buffer. After washing, the protein was eluted from the column with 300 mM NaH(2)PO(4), pH 5.1, 1 mM EDTA buffer (Zhang and Van Etten, 1990). Protein fractions of greater than 99% purity (based on overloaded SDS gel electrophoresis patterns) were concentrated to 5-10 mg/ml using a Filtron microconcentrator. These samples were chromatographed on a Sephadex G-25 size exclusion column (1.5 times 4 cm) preequilibrated with 0.01 M Tris, 0.14 M NaCl, pH 7.0. Fractions were collected, assayed for inorganic phosphate content (Black and Jones, 1983), and those containing less than 20 µM inorganic phosphate were again concentrated and loaded on a freshly pre-equilibrated Sephadex G-25 size exclusion column. Fractions which contained less than 6 µM inorganic phosphate were concentrated and used in this study. Upon final dilution of the sample the inorganic phosphate concentration drops to less than 1 µM, which is well below the 2 mMK(i) for phosphate.

Enzymatic Activity

Phosphatase activity was determined at 37 °C by using 10 mMp-nitrophenyl phosphate (pNPP) as a substrate in 100 mM sodium acetate buffer, 0.08 M NaCl, pH 5 (Wo et al., 1992). Protein concentrations were determined either by the Lowry method (stock enzyme spectrophotometric solutions) or by measurement at 280 nm using extinction coefficients calculated by the method of Gill and von Hippel(1989) of 18,740, 13,050, 13,050, and 7,360 M cm, for WT, W39F, W49F, and W39,49F BPTP, respectively.

Circular Dichroism Spectral Measurements

The CD spectra of 7 µM WT BPTP and mutant proteins in 10 mM sodium phosphate, pH 5.0, were measured over the range of 190-240 nm in a 1-mm cell using a Jasco model J600 CD Spectropolarimeter.

Steady-state Kinetics

Michaelis-Menten parameters for WT BPTP and mutants were determined using pNPP in 100 mM sodium acetate buffer, 1 mM EDTA, 0.08 M NaCl, pH 5. Nine different pNPP concentrations ranging from 0.1 to 4.1 mM were used and each measurement was performed in triplicate. K(m) and V(max) values were determined by fitting the data directly to the Michaelis-Menten equation and minimizing ^2 using a macro written within Quattro Pro.

Quantum Yield

L-Tryptophan at 25 °C (Q = 0.14) was used as a reference for the quantum yield determinations of WT BPTP and the single tryptophan mutant proteins (Kirby and Steiner, 1970). Typical optical densities of the protein and L-tryptophan were in the range of 0.2 to 0.4 and were determined at a wavelength of 295 nm using a Hewlett Packard model 8450A spectrophotometer. Quantum yield values were calculated using , where and are the quantum yields, I and I are the integrated intensities of the emitted spectra

over a range of 300 to 400 nm (corrected) with excitation at 295 nm, and OD and OD are the optical densities of the given solutions at 295 nm. Determinations were performed in triplicate and the values were recorded as averages.

Determination of Intramolecular Distances of Trp or Trp to the Active Site

Samples of mutant proteins W39F, W49F, and W39,49F were diluted to concentrations ranging from 1 to 2 mg/ml in 10 mM diethylmalonate, pH 7.5, containing 75 mM succinate, 0.15 M NaCl, and 1 mM EDTA. IAEDANS was added to each sample to a concentration of 7 mM. The samples were shielded from light for 1 h at room temperature. At this time, 95% of the enzyme was inactivated. Excess modification agent was removed by passing each sample through a Sephadex G-25 column pre-equilibrated with 0.01 M Tris, 0.14 M NaCl, pH 7.0. Fractions containing the greatest amount of protein as determined by their A were diluted with 0.01 M Tris, 0.14 M NaCl, pH 7.0, to obtain an absorbance of 0.05-0.10 at a wavelength of 295 nm. The L-tryptophan solution displayed an absorbance of 0.06 at 295 nm. The efficiency of energy transfer (E) is related to the intramolecular distance R between the donor and acceptor by , where R(o) is the Förster distance of 22 Å (Wu and Brand, 1994).

Determination of Fluorescence Lifetimes of WT BPTP and Single Tryptophan Mutants

Protein solutions were typically prepared to have absorbances of 0.2-0.3 at 295 nm. Analysis of data for temporal information was performed using an in-house non-linear least-squares Marquardt algorithm (Demas, 1983; O'Connor and Phillips, 1984).

Quenching Experiments

Quenching experiments were performed using steady-state emission spectral techniques. Stock quenching solutions of acrylamide, KI, and CsCl were 5.0 M, 2.5 M, and 2.5 M, respectively. The buffering system used for the acrylamide experiments was 0.01 M Tris, 0.14 M NaCl, pH 7. The ionic strength of samples quenched by KI or CsCl was brought to 0.15 M by addition of sufficient 2.5 M KCl in 0.01 M Tris, pH 7. The potassium iodide stock solution contained 0.1 mM sodium thiosulfate to prevent formation of I(3). For dynamic quenching, sample solutions were contained in a NSG T-52 dual pathlength (1 cm times 4 mm) cell.

Aliquots of the WT and mutant protein solutions (5 mg/ml) were diluted using appropriate proportions of quenching stock solution, buffer, and if necessary KCl solution to obtain a range of quenching concentrations. Dilution corrections were made unnecessary since enzyme concentrations were identical upon dilution. Corrections for absorptive screening by acrylamide ( = 0.25 M cm) were made as described previously by Eftink and Ghiron(1976). Quenching results at 328 and 350 nm were plotted using intensity ratios.

The pH Dependence of Tryptophan Lifetime Components for W39F, W49F, and WT BPTP

Stock solutions of enzyme (5 mg/ml) in water were diluted using the following buffers: pH 3.5-4.0, 100 mM dimethylglutarate; pH 4.5-5.5, 100 mM sodium acetate; pH 6.0-7.0, Bis-Tris; pH 7.0-8.5, 100 mM Tris. The ionic strength of each buffer mixture was adjusted to 0.15 M by addition of a sufficient amount of NaCl as determined from ionic strength calculations. The long and short lifetime components were plotted independently as a function of solution pH.


RESULTS

Characterization of WT BPTP, W39F, W49F, and W39,49F

The mutant proteins were readily expressed using procedures developed earlier for the expression of WT BPTP (Wo et al., 1992; Davis et al., 1994). SDS-polyacrylamide gel electrophoresis analysis using a 10% gel, with Coomassie Brilliant Blue R-250 staining, showed that each enzyme (25-35 µg/lane) consisted of the expected single band of M(r) 18,000. The Michaelis-Menten parameters of WT BPTP and the single and double tryptophan mutant proteins measured after a cycle of denaturation/renaturation are summarized in Table 1. For comparison, WT BPTP and the W49F mutant protein displayed V(max) values of 106 and 85 µmol/(min-mg), respectively, before denaturation/renaturation. These values are effectively identical to the results obtained for the respective proteins after denaturation/renaturation (Table 1), consistent with the fully reversible denaturation of the wild type enzyme isolated from tissue (Zhang, 1990). Thus, replacement of tryptophan by phenylalanine at position 49 caused little change in enzymatic activity. In contrast, the initially isolated preparations of the W39F and W39,49F proteins exhibited reduced specific activities of 14 and 20 µmol/(min-mg), respectively, before denaturation and renaturation. However, following denaturation and renaturation of W39F and W39,49F at 4 °C, the activities of these two mutant proteins were in fact only modestly reduced compared to those of WT BPTP and W49F (Table 1). Further analysis by CD confirmed that the secondary structures of these enzymes did not appear to be significantly altered by mutation. Highly similar log k/K(m) values for WT BPTP and the tryptophan mutants are consistent with the conclusion that the mechanism by which WT BPTP proceeds is conserved in the tryptophan mutant proteins. The renatured proteins, W39F and W39,49F were found to be as stable as WT BPTP during the time period at room temperature that was needed to complete spectral measurements.



The extinction coefficients of wild type and mutant proteins are given in Table 2. Extinction coefficients measured at 295 nm were significantly decreased from those at 280 nm. The excitation maxima of the proteins were nearly identical, ranging from 277 to 280 nm. The steady-state emission spectra were obtained for WT BPTP and the mutant proteins W39F and W49F at an excitation wavelength of 295 nm and are corrected for any influence of tyrosyl residues by subtraction of the W39,49F fluorescence spectra (Fig. 1). By summing the fluorescence intensities of the single tryptophan mutants, a spectrum that closely resembles that of WT BPTP was generated (Fig. 1). WT BPTP has an emission maximum at about 342 nm, typical of the situation for most native proteins, which have emission maxima in the range of 331 to 343 nm. Trp is slightly red shifted. In contrast, Trp shows a dramatic blue shift to an emission maximum of about 325 nm. Quantum yields of WT BPTP and mutants are summarized in Table 2. The quantum yield of Trp is considerably higher than of Trp and when the two values are summed the total is equivalent to that observed for WT BPTP.




Figure 1: Emission spectra of WT BPTP and the mutant proteins W39F and W49F at 20 °C in 0.01 M Tris buffer, 0.14 M NaCl, pH 7; excitation was at 295 nm. Each spectrum is corrected for tyrosine interference by subtraction of the fluorescence spectrum of the double mutant protein W39,49F. The composite spectrum resulting from the sum of the contributions due to W39F and W49F is indicated by the dashed line.



Determination of Intramolecular Distances of Trp or Trp to the Active Site

Using the respective AEDANS-labeled single tryptophan mutant proteins, fluorescence energy transfer was observed for both tryptophan residues. No energy transfer was observed for the AEDANS-labeled W39,49F mutant protein. The transfer efficiency of donor Trp (measured with mutant protein W49F), or Trp (measured with W39F), to a single AEDANS acceptor located at the active site of each mutant protein was used to estimate the intramolecular distance between the tryptophan residue and the active site of the enzyme. The calculated distances of Trp and Trp to the active site are thus 21 and 9 Å. The intramolecular distance calculated for Trp to the active site may be biased by other interactions or energy transfer mechanisms (Wu and Brand, 1994).

Determination of Tryptophan Lifetime Components for WT BPTP, W39F, and W49F

The non-linear least-squares Marquardt algorithm was used to examine fluorescence decays from WT BPTP. Goodness of fit parameters such as ^2 and weighted residuals allow an estimate of the number of lifetime terms appropriate for consideration in the spectroscopic model. Fig. 2A shows the fit to the data when using one exponential, while Fig. 2B shows the results for an equation with two exponential terms. With a one-term model, ^2 was 32.2 and the lifetime 6.59 ns. A visual examination of the poor quality of the fit and the non-random distribution of the residuals indicates that a single decay does not properly account for the experimental result. With a two-term model, ^2 was 1.14 with lifetimes of 1.62 (alpha(1) = 0.51) and 7.54 (alpha(2) = 0.49) ns. Visual examination of the quality of the fit finds it quite good except at the peak of the decay, with the remaining residuals randomly distributed as a function of time. A model employing three terms yielded a ^2 of 1.08 with lifetimes of 1.16, 2.61, and 7.68 ns. The only apparent improvements were the slight lowering of ^2 and a better fit near the peak of the decay. A final model employing four terms yielded the same value of 1.08 for ^2 with lifetimes of 1.28, 3.14, 7.50, and 8.02 ns. The visual fit was no better than the three-term model. When the mathematical analysis was repeated with replicate decays, the two component model maintained nearly the same lifetime values. In contrast, the lifetimes produced by the three and four component models varied to a much greater extent. Consequently, a two component fit was adopted as a working model, with one decay tentatively assigned to each of the tryptophan residues. Using this model, decays were recorded as a function of emission wavelength. Two trends were noted. Both decays displayed a monotonic increase in lifetime progressing from 315 to 395 nm, the fast decay varied from a low of 1.25 ns to a high of 2.75 ns, and the slow decay varied from a low of 5 ns to a high of 7.5 ns. The second trend involved the amplitudes. At 315 nm the short lifetime was responsible for 82% of the initial intensity and monotonically decreased to longer wavelengths, while at 385 nm the long lifetime was responsible for 72% of the initial intensity and monotonically decreased toward shorter wavelengths. These observed trends were inconsistent with the model. It was expected that the total amplitude would deconvolve, on the basis of lifetimes, into components resembling spectra for each of the 2 tryptophan residues. On the basis of these results as well as some quenching behavior to be discussed in the next section, it was decided to prepare and examine the spectral properties of the three mutant proteins W39F, W49F, and W39,49F.


Figure 2: A, fluorescence decay spectrum of WT BPTP overlaid with a calculated single exponential decay of 6.6 ns. B, the same spectrum overlaid with calculated two component exponential decay where (1) = 1.62 ns and (2) = 7.54 ns. In both cases the decay spectrum of WT BPTP is characterized by a higher amplitude at the peak apex than either one of the calculated decay spectra. Excitation was at 295 nm, and the spectra for WT BPTP were obtained at 350 nm.



The fluorescence lifetime data for W39F, W49F, and WT BPTP are summarized in Table 3. The most notable observation is the dual decay associated with each tryptophan residue. The lifetimes and amplitudes remain constant across the two individual emission spectra. The Marquardt algorithm was unable to successfully resolve the four fluorescence lifetimes expected to be present in the case of WT BPTP. That is, the two component, three component and four component models all produced lifetimes that were weighted averages of the actual decays. Four of the two component fits are shown in Table 3to demonstrate this effect.



To be certain that the fluorescence behavior of WT BPTP is properly described by combining the properties of W39F and W49F, the following numerical analysis was performed. The individual decay data at 350 nm for W39F and W49F were added in proportion to their relative strength given in Fig. 1. Treating this combined decay with the Marquardt algorithm yielded a two-component decay with lifetimes (1.46 and 7.49 ns) and amplitudes (0.66 and 0.33) that are statistically indistinguishable from those obtained directly from WT protein.

Steady-state Quenching Experiments on BPTP and Single Tryptophan Mutants

Quenching experiments are commonly performed with tryptophan to probe the extent of exposure to the exterior solvent. In the present studies, the quenching ability of neutral acrylamide, anionic iodide, and cationic cesium were compared. Care was exercised with acrylamide since it is known to exhibit static quenching (ground state formation of a non-fluorescent complex) at higher concentrations. The presence of such quenching is characterized by an upward curvature of the Stern-Volmer plot (Eftink and Ghiron, 1981). The Stern-Volmer plots for acrylamide, KI, and CsCl quenching are shown in Fig. 3for the two mutant proteins, W49F (328 nm) and W39F (350 nm). WT BPTP and all mutant proteins were found to retain full activity when exposed to individual quenching agents within the range of concentrations studied. For the concentrations used in Fig. 3, the Stern-Volmer plots show a linear relationship between fluorescence intensity and quenching reagent concentration. Only at very high concentrations of acrylamide did the slopes deviate from linearity (data not shown). As a control, quenching experiments were performed using yeast 3-phosphoglycerate kinase, and the results were in good agreement with those previously published (Stryjewski and Wasylewski, 1986; Eftink, et al., 1987; Dryden and Pain, 1989).


Figure 3: Stern-Volmer plots for acrylamide (box, W39F; , W49F), iodide (up triangle, W39F; , W49F), and cesium (down triangle, W39F; , W49F) at 20 °C and pH 7; excitation was at 295 nm.



The computed bimolecular quenching constants for W39F and W49F are summarized in Table 4. The k(q) obtained for Trp (in W39F) are 1.5-3 times larger than those observed for Trp (in W49F). The magnitudes of k(q) for W39F using acrylamide and iodide ion clearly demonstrate that Trp is exposed to a much greater extent than is Trp. Quenching studies of Trp and 49 with Cs suggest that Trp was again quenched to a higher extent. Since the quenching efficiencies of acrylamide and iodide are 1.0 and cesium is 0.2, an unambiguous interpretation of cesium quenching data could not be made. In order to compare further these three quenching agents the ratio of k /k for protein to indole was used. These ratios were 0.45, 0.50, and 0.43 for acrylamide, iodide, and cesium, respectively. These data suggest that the ability of Csto quench Trpdid not appear to be enhanced compared to the neutral and anionic reagents. This would indicate a lack of neighboring anionic residues. Since the degree to which Trpis exposed is primarily dependent on the fluctuations of protein structure, the ratio of apparent quenching constants is no longer diffusionally dependent and thus cannot be used to infer local ionic structure.



Steady-state Stern-Volmer studies on WT BPTP require that data interpretation take into account the different quenching rates of each tryptophan. To this end, effective values of K and k(q) are necessary. The effective Stern-Volmer and bimolecular quenching constants for WT BPTP are shown in Table 4. Values of k(q) obtained for WT BPTP at 328 and 350 nm could mislead one to conclude that both Trp and Trp are essentially equally exposed to the solvent (assuming that the tryptophan fluorescence of each residue is homogeneous). This incorrect result is due to the fact that the intensity from the short-lived tryptophan is dominated by that from the long-lived component, thus skewing the intensity results to a higher F(O)/F value at 328 nm than would be expected on the basis of its smaller quenching constant.

The pH Dependence of Tryptophan Lifetime Components in WT BPTP and Single Tryptophan Mutants

Time-correlated single photon counting profiles of WT BPTP and the mutant protein W39F yielded some information concerning the influence of ionizable amino acid residues on quenching accessibility and the natural quenched states of tryptophans 39 and 49. Comparison of the pH titration of WT BPTP and W39F suggests that the fluorescence of Trp is affected by the protonation state of an acidic residue with an apparent pK(a) of 4. In contrast, the fluorescence of the mutant protein W49F shows no change over the pH range 3.5-8.


DISCUSSION

Proteins containing more than one tryptophan exhibit multiple lifetimes due to the large number of configurations and microenvironments that the tryptophan residues may experience. With the aid of single and double tryptophan mutant proteins, the present study has examined the individual fluorescence characteristics of Trp and Trp in BPTP. Comparison of lifetime and steady-state fluorescence results of BPTP and its single tryptophan mutant proteins raises significant questions as to the accuracy with which the fluorescence of 2 such tryptophans could be resolved without the use of single tryptophan mutants.

The fluorescence decay of WT BPTP consists of a short component that is primarily blue shifted and a long component emitting at lower energy. It was initially assumed that since the environments of these tryptophans were substantially different, each component described only 1 of the tryptophan residues. From intensity profiles we were able to attribute the amount of fluorescence apparently due to each residue at particular wavelengths. The k(q) values for the short and long components of WT BPTP were found to be essentially equivalent which would indicate that the accessibilities of Trp and Trp to quenching agent were only slightly different. However, upon analysis of the single tryptophan mutant proteins, this was clearly found not to be the case. Both single tryptophan mutant proteins were found to exhibit biexponential fluorescence decay, and, therefore, the decay of WT BPTP would be more accurately described as a quaternary exponential. Erroneous quenching results arise from the fact that Trp possesses not only a 7.18-ns component, but also a 2.05-ns component that makes a fluorescence contribution to the faster decaying tryptophan of WT BPTP. Therefore, the quenching effects on the exterior tryptophan dominated the Stern-Volmer data analysis and appeared as a property of the buried residue. This conclusion is also confirmed by fluorescence analysis of Trp (measured with the mutant protein W49F), which possessed lifetimes of 3.36 and 1.04 ns, and which was inherently quenched to a much greater extent than the short-lived component observed at 328 nm for WT BPTP.

Lack of any inter-tryptophanyl homotransfer of energy is supported by the agreement of the sum of the quantum yields of Trp and Trp with that observed for WT BPTP. Additionally, the fluorescence emission spectrum of WT BPTP is identical to that of the combined fluorescence spectra due to Trp and Trp, as measured with the single tryptophan mutant proteins.

The reduced activities of the W39F mutant proteins that were observed before denaturation-renaturation are attributed to a change in protein stability or alterations in protein folding, since the purification and assay procedures were similar to that for WT and W49F. Typically, 60% of tryptophan side chains are involved in hydrophobic binding and contribute -0.72 to -1.4 kcal mol of energy to protein stability (Hart et al., 1993; Dill, 1990). Consistent with recent NMR and x-ray structural data (Logan et al., 1994; Zhang, et al., 1994; Su et al., 1994) the present fluorescence steady-state and lifetime studies show definitively that Trp is situated in a highly hydrophobic core region of the protein. Trp is slightly red shifted to 328 nm and possesses a quantum yield of 0.05, also consistent with its localization within a hydrophobic pocket. The dramatically low quantum yield suggests that the indole moiety may interact with the peptide backbone (Burstein et al., 1973; Ricci and Nesta, 1976). Quenching of Trp reveals that it is partially accessible to quenching agents. (Note, however, that the short lifetime already indicates significant quenching, possibly by the peptide backbone, and, therefore, the effect of additional quenching by external agents would be expected to be difficult to detect). The calculated intramolecular distance of 21 Å between Trp and the active site shows that Trp is unlikely to play a direct role in affecting the enzymatic activity of BPTP. Instead, it presumably contributes to the stability of the protein by participation in the hydrophobic core. Minor perturbations in structure are not uncommon in proteins that possess mutated residues within the interior core of their structure (Leatherbarrow and Fersht, 1987). The crystal structures of these and other mutants are under study in order to directly measure these changes.

Trp is moderately red shifted (to an emission maximum of 350 nm) and has a quantum yield of 0.18, which are typical characteristics of tryptophan residues located on the exterior of proteins. Quenching studies confirm this result, with large k(q) values in the range of 3 times 10M s for iodide and acrylamide. Quenching by cesium does not indicate anionic charges within the immediate vicinity, but pH studies indicate the presence of an ionized acidic residue proximal to Trp which is shielded from approach by quenching agents. This residue is presumably aspartic acid 48. Trp is located on the exterior of the protein and is fully exposed to quenching agents. Energy transfer studies indicate that Trp is within 10 Å of the active site, consistent with recent structural data (Logan et al., 1994; Zhang et al., 1994). Young et al.(1994) have recently suggested that ``surface hydrophobicity can be used to identify regions of a protein's surface most likely to interact with a binding ligand.'' It is very likely that this surface tryptophan is part of such a surface cluster.

In conclusion, our results provide information about the properties and locations of the tryptophan residues of BPTP and clearly demonstrate the necessity of using single tryptophan mutants to characterize the fluorescence decay of WT BPTP. Work is presently in progress involving the use of these tryptophan residues as probes to monitor enzyme conformational changes.


FOOTNOTES

*
This work was supported by United States Department of Health and Human Services Grant GM 27003 and by National Science Foundation Grant CHE-8822878. 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 should be addressed. Fax: 317-494-0239.

(^1)
The abbreviations used are: PTPases, protein tyrosine phosphatases; BPTP, bovine phosphotyrosyl protein phosphatase; WT, wild type; pNPP, p-nitrophenyl phosphate; IAEDANS, 5-[[[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid; AEDANS, 5-[[(acetylamino)-ethyl]amino]naphthalene-1-sulfonic acid.


ACKNOWLEDGEMENTS

We acknowledge Keith D. Grinstead Jr. for assistance with preliminary aspects of this research, Dr. June Davis for producing one of the tryptophan mutants, Zhongtao Zhang for help with the IAEDANS labeling experiment, Marie Zhang for comments on the x-ray crystal structure, and Professor Frank Bright for several helpful discussions.


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