(Received for publication, August 10, 1994; and in revised form, December 16, 1994)
From the
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
.
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) ()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.
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 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
PO
, 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
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
for phosphate.
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.
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
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.
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 = 1.62 ns and
= 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.
Figure 3:
Stern-Volmer plots for acrylamide
(, W39F;
, W49F), iodide (
, W39F;
, W49F),
and cesium (
, 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 obtained for Trp
(in W39F) are 1.5-3 times
larger than those observed for Trp
(in W49F). The
magnitudes of k
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
Cs
to quench Trp
did 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 Trp
is 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
are necessary. The effective Stern-Volmer and bimolecular
quenching constants for WT BPTP are shown in Table 4. Values of k
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
/F value at 328 nm than would be expected on the basis of its smaller
quenching constant.
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 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
values in the range of 3
10
M
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.