(Received for publication, March 2, 1995; and in revised form, April 7, 1995)
From the
Early studies suggested that the Escherichia coli tryptophan synthase The The Matthews group has
employed several singly and doubly altered To utilize our collection of 70-80 trpA mutants(18, 19) , which exhibit a broad spectrum
of thermal instability, we have constructed several wild-type-like host
proteins into which we could insert these mutational changes. The goal
in preparing such wild-type-like host
The mutagenesis/sequencing vector for Phe
The tryptophan-containing All
have expected increases in UV absorbance. Mutant
Figure 1:
Fluorescence spectra,
emission
Figure 2:
Urea-induced unfolding of the wild-type (
Figure 3:
The fractional unfolding, F
A quantitative comparison of these
equilibrium data was made by statistically modeling each data set for a
two-step, three-state unfolding model, following the classical
treatment for multistep unfolding processes in which there are
populated intermediates:
where K this relationship can be reformulated as:
where K and
where m
As pointed out by Beasty et al.(25) and Cupo and Pace(27) , because of potential
errors due to the large extrapolation required to determine
The apparent wild-type enzymatic and urea stability (as
monitored by UV absorbance) properties of these tryptophan-containing
-subunit unfolded in a two-step process
in which there was a stable intermediate composed of a native
-1
folding unit (residues 1-188) and a completely unfolded
-2
folding unit (residues 189-268). More recent evidence has
indicated that such a structure for the intermediate seems unlikely. In
this report, single Trp residues (absent in the wild-type
-subunit) are substituted separately for Phe residues at positions
139 (in
-1) and 258 (in
-2) to produce the F139W, F258W, and
F139W/F258W mutant
-subunits. The UV absorbance and fluorescence
properties of the F139W/F258W double mutant are identical with those of
equimolar mixtures of the single mutants, suggesting that the Trp
residue at each position can independently report the behavior of its
respective folding unit. Each mutant
-subunit is wild-type
enzymatically, and when UV absorbance is monitored, the urea-induced
unfolding of the three tryptophan-containing
-subunits is
virtually identical to the wild-type protein. These wild-type
properties make these proteins attractive candidates for a fluorescence
examination of the behavior of the individual folding units and the
structure of potential intermediate(s) and as host proteins for the
insertion of our existing destabilizing and/or stabilizing mutational
alterations.
-subunit of the Escherichia coli and/or Salmonella typhimurium tryptophan synthase has long been used
as a model to explore the stability and unfolding and folding
properties of proteins and polypeptides. The
-subunit consists of
a single polypeptide chain of 268 amino acid residues and contains no
prosthetic groups. Structurally(1) , it is a member of the
/
barrel family, belonging to a subgroup that includes
phosphoribosylanthranilate isomerase, indoleglycerolphosphate synthase,
and triosephosphate isomerase (2) and is composed of 11
-helices and 8
-strands
(
). Studies on the unfolding
properties of the E. coli protein by urea and/or guanidine
were initiated more than a decade ago by the
Yutani(3, 4) , Matthews(5) , and Miles (6, 7) groups. The unfolding behavior indicated the
presence of a partially unfolded, stable intermediate during the
unfolding process. This conclusion was supported by the finding that
there was a hypersensitive trypsin site at
Arg
(6) , subsequently determined to be located
within a region (residues 178-191) that is highly mobile in the
crystal structure(1) . Cleavage at this site yields two
fragments, an N-terminal
fragment
(
-1) composed of
-strands 1-6 of the barrel structure
and a C-terminal
fragment (
-2)
containing the remaining barrel
-strands 7 and 8. In guanidine,
each of these can unfold and refold independently, and when combined,
the
-2 folding unit unfolds prior to
-1. These results led
initially to the notion that a stable unfolding intermediate existed
containing a native
-1 unit and an essentially completely unfolded
-2 unit. In a subsequent extensive analysis by Matthews'
groups(8, 9, 10, 11, 12) ,
a refolding pathway was elaborated, which was generally consistent with
such a structural intermediate. Although the existence of separate
folding units appears valid, more recent studies by the Matthews (13, 14, 15) and Yutani groups (16, 17) have substantially modified this picture of
the structure of the unfolding intermediate.
-subunits in examining
temperature effects on urea denaturation by UV and proton NMR methods.
It was concluded that the
-2 unit can assume an organized
structure in the intermediate state. The total structure is proposed to
be a loosened form of the barrel structure in which
-strand 6 (in
-1) and
-strand 7 (in
-2) have altered packing, although
they continue to interact. Not inconsistent with these results, the
Yutani group, utilizing calorimetry, CD, and tyrosine fluorescence, has
described the equilibrium intermediate structure as a molten globule
form.
-subunits was to be able to
monitor separately the behavior of the putative individual
-1 and
-2 folding units while still utilizing the intact protein. To this
end, we have substituted a single tryptophan residue within the
-1
folding region (at position 139) in one such host protein and one
tryptophan residue within the
-2 folding region (at position 258)
in another. The normal
-subunit contains no tryptophan. We report
here the suitability (i.e. the spectral properties and
wild-type behavior) of these tryptophan-containing
-subunits to
serve as host proteins and to provide additional information regarding
both the unfolding and refolding pathways for each folding unit and the
structure of potential intermediate(s).
Chemicals and Enzymes
All chemicals
(reagent or ultrapure grade) and enzymes for mutagenesis and sequencing
were obtained from Sigma or New England Biolabs. Ultrapure urea was
from Life Technologies, Inc. and was used without further purification.
Urea concentrations were determined by refractive index measurements.Mutagenesis
Oligonucleotides were
synthesized on a Milligen Biosearch model 8700 DNA synthesizer and
purified by reverse phase chromatography on Waters oligo-pak columns.
For the Phe
Trp
change, the
oligonucleotide 5`-GAGTCCGCGCCCTGGCGGCAGGCCGCG-3` was used. The
sequence alterations from the wild-type sequence (TTCCGG) are underlined and represent those that encode
Phe
-Arg
in the
-subunit. There is a
3-base pair change, which results in the Phe
Trp substitution
only but introduces an additional BglI site into the trpA gene that can be used for the initial mutant selection. For the
Phe
Trp
change, the oligonucleotide
5`-GGCGCGACTGAAAGTTTGGGTCCAACCGATGAAAGCGGCG-3` was used. The changes
from the wild-type sequence (TTTGTA), again underlined, result
in a Phe
Trp substitution only, but in this case there is the
loss of an RsaI site in trpA, and this is used for
initial mutant selection. The sequence alterations were confirmed by
DNA sequencing.
mutagenesis was similar to those described before (18, 20) except that a trpABsaHIXhoI sequence (encoding residues
124-177 in the
-subunit) from ptactrpAMK (20) was
inserted immediately 3`-distal to the GC clamp. The
mutagenesis/sequencing vector for Phe
mutagenesis was
pGCtrpAVI as described before(20) . The procedures for
oligonucleotide-directed mutagenesis and confirmation of the sequence
change by restriction nuclease/DNA sequencing are described elsewhere (20) . The mutagenized fragments containing the altered
sequences at positions 139 and 258 were subcloned as described before (20) into modified expression vectors of pCATtrpA and
ptactrpAMK, respectively. These subclonings yielded expression vectors
pCATtrpF139W and ptactrpFW258, which encode the F139W and F258W mutant
-subunits. The double mutant, F139W/F258W, was constructed by
substituting the trpABglII-XhoI fragment
(encoding residues 1-177) from pCAT trpF139W for the
corresponding wild-type fragment in ptactrpF258W to yield
ptactrpF139W/F258W.
The - and
-Subunit
Purifications
-subunits (wild type, F139W, F258W,
and F139W/F258W) were purified from E. coli strain RB797
containing, respectively, ptactrpAMK, pCATtrpF139W, ptactrpF258W, and
ptactrpF139W/F258W. Growth conditions and
-subunit purification
protocols were identical for all and have been described
before(20) . The
-subunit was obtained also as previously
detailed(20) .
Protein and Activity Assays
Wild-type
-subunit concentrations were determined using the molar extinction
coefficients at 278 nm of 12,700 cm
. Concentrations
of the tryptophan-containing
-subunits were determined initially
by the microbiuret assay (21) and by comparison with the
microbiuret results with the wild-type protein; the molar extinction
coefficients at 278 nm were estimated for the F139W, F258W, and
F139W/F258W
-subunits. The concentration of the
-subunit was
estimated with the E
of 6.5(22) .
The enzymatic activities of the
-subunits in the tryptophan
synthase
- and
-reactions were performed according to
Lim et al.(23) ; the
-reaction assays were
according to Sarkar et al.(20) . Values of k
for the wild-type
-subunit (in the
presence of the
-subunit) in the
-,
-, and
-reaction assays were 0.18 s
, 11.9
s
, and 6.21 s
, respectively.
Spectrophotometric Measurements
All
measurements were recorded at ambient temperatures, invariably
23-24 °C. The buffer in all experiments is 10 mM potassium phosphate (pH 7.8), 0.2 mM disodium EDTA, and 1
mM -mercaptoethanol. Corrections were made for buffer and
denaturant. UV absorbance measurements for equilibrium studies were
made with a Perkin-Elmer dual-beam
3A spectrophotometer. A
Perkin-Elmer luminescence/fluorescence spectrometer model LS50B was
used for the fluorescence spectral analysis. The experimental data for
the unfolding experiments presented here represent the average of three
to six experiments. The standard deviation for each measurement is
±5-10% or less. The data analysis program is a standard
non-linear regression utilizing the Levenberg-Marquardt method as
described in Press et al.(24) .
Enzymatic and Spectroscopic Properties of the
Tryptophan-containing
The goal in selecting
residue sites within the -Subunits
-subunit at which to insert tryptophan
residues to produce suitable wild-type host proteins was that the
substitution would not itself lead to major changes in function and/or
stability to denaturants. The selection of Phe
(the
second residue in helix 4) in the
-1 folding unit and Phe
(a middle residue in helix 9) in the
-2 folding unit was
based on a variety of considerations such as lack of conservation at
the site, relative residue size, hydrophobicity, secondary
structure-forming tendencies, and solvent accessibility. Despite such
considerations, it was difficult to predict accurately the effect of
tryptophan substitutions at these sites. It was important, therefore,
at the outset to characterize some of their enzymatic spectroscopic and
urea stability properties.
-subunits
were examined for their enzymatic activity in the three tryptophan
synthase reactions: the
-,
-, and
-reactions. The
-reaction measures the functional integrity of the
-active
site with respect to the catalysis of the aldolysis of
indole-3-glycerol phosphate to indole and D-glyceraldehyde-3-phosphate. The
-reaction measures the
structural capacity of an
-subunit to (a) allow access of
indole from the solvent to the
-active site; (b) provide
the proper alignment of the intramolecular indole channel between the
subunits; and (c) bring about proper conformational changes in
the
-subunit to activate its catalysis of indole plus L-serine to L-tryptophan. The
-reaction
measures the ability of an
-subunit to (a) catalyze an
accelerated rate of aldolysis of indole-3-glycerol phosphate at the
-active site; (b) transfer the enzymatically formed
indole intramolecularly to the
-active site; and (c)
undergo conformational changes induced by the
-subunit (following
binding of L-serine to the
-active site) that lead to the
increased rate of indole-3-glycerol phosphate utilization.
-Subunits F139W, F258W, and F139W/F258W had relative specific
activities, respectively, of 96, 89, and 105% that of the wild-type
-subunit in the
-reaction; 98, 92, and 106% that of the
wild-type
-subunit in the
-reaction; and 102, 104, and 100%
that of the wild-type
-subunit in the
-reaction.
-subunits F139W,
F258W, and F139W/F258W have estimated molar extinction coefficients at
278 nm of 16,700 cm
, 16,690 cm
,
and 20,800 cm
, respectively, compared to that of
12,700 cm
of the wild-type
-subunit. These
increases in molar extinction values (above that of the wild-type
-subunit) of 4000 cm
, 3990
cm
, and 8100 cm
, respectively,
are consistent with the introduction of either one or two tryptophan
residues. A molar extinction value of 4400 cm
for L-tryptophan is obtained under similar conditions. The
fluorescence behavior of the tryptophan-containing mutants is shown in Fig. 1. All exhibit an emission
shift to
lower wavelengths and fluorescence intensity increases of varying
magnitude (relative to free tryptophan), suggesting that the tryptophan
residues are buried in less polar environments within the respective
proteins. The spectrum and relative fluorescence intensities of a
equimolar mixture of F139W and F258W are virtually identical to the
double mutant.
, and the relative fluorescence intensities
for L-tryptophan and the F139W, F258W, and F139W/F258W
-subunits (solid lines) and a mixture of F139W plus F258W
-subunits (dashed lines). All concentrations were 5
µM, the excitation wavelength was 295 nm, and the scan
rate was 120 nm/min with slit widths of 5
nm.
Urea Stabilities of the Tryptophan-containing
For comparative purposes, the urea stability
behavior of the wild-type and mutant -Subunits
-subunits was monitored by UV
absorbance changes according to the methods of Matthews and co-workers (5, 25) . Fig. 2shows the absorbance changes
upon urea addition to the wild-type and mutant
-subunits; the
differences reflect the differences in their respective extinction
coefficients. Applying corrections for these differences and assuming
that the absorbance changes at 6 M urea represent essentially
complete unfolding(5, 25) , the fractional unfolding versus urea concentrations is given in Fig. 3, A-D, where f
= (
-
)/(
-
),
is the observed extinction
coefficient, and
and
are the extinction coefficients of the native and unfolded
forms. All commence unfolding at 2.2-2.5 M urea and are
essentially completely unfolded at 5-5.5 M urea.
-Subunit F139W appears virtually identical to the wild-type
-subunit;
-subunits F258W and F139W/F258W appear to be only
slightly more stable. In addition, the unfolding transitions for all
appear to be completely reversible (Fig. 3, A-D, opencircles).
), F139W (
), F258W (
), and
F139W/F258W (
)
-subunits. The protein
concentrations were 35 µM.
, versus urea concentration for
wild-type (A), F139W (B), F258W (C), and
F139W/F258W (D)
-subunits (solid circles). The solidline is that for a two-step, three-state
unfolding transition. The opencircles represent the
calculated F
observed upon dilution from 6 M urea to the indicated urea concentration. PanelsA`-D` present the fractions of native (f),
intermediate (f), and unfolded (f) forms during
unfolding of wild-type (A`), F139W (B`), F258W (C`), and F139W/F258W (D`)
-subunits.
is the apparent equilibrium
constant, K
is the product of the K
values for the individual steps, and Z
is the fractional change in the
measured parameter for the product of each step. For the case of a
three-state, two-step model as suggested for the
-subunit, namely
and K
are the
equilibrium constants for the N
I, I
U transitions, respectively, and Z = (
-
)/(
-
) where
is the
extinction coefficient of the intermediate form. The results of these
statistical analyses for the wild-type and tryptophan-containing
-subunits are shown by the solidlines on Fig. 3, A-D. For all the
-subunits
(wild-type, F139W, F258W, and F139W/F258W), the fit to a two-step model
appears to fit the data well. Assuming a linear dependence of free
energy on urea concentration, the free energy of unfolding in the
absence of denaturant, G
2
, was estimated for
each step according to Pace(26) :
, m
(the
slopes) are the cooperativity factors for each plot, and C is
the urea concentration. The
G
2
, m, and Z values for the four
-subunits are given
in Table 1. The values for the wild-type
-subunit compare
favorably to those obtained by Beasty et al.(25) and
are shown in parentheses. Mutant
-subunit differences from the
wild-type (at zero urea concentrations) in
G
2
and
G
2
vary between -0.6 and
-1.4 kcal/mol.
G
2
and the legitimacy of a linear
relationship in this extrapolated range, a more appropriate comparison
of stability would be at a urea concentration other than zero. A
suitable reference point for comparison would be the urea concentration
at the midpoints of the two transitions (N
I, I
U) for the reference wild-type
-subunit, at which
G
and
G
are
zero. Utilizing the Z values obtained, the relative
proportions of native (f
), intermediate (f
), and unfolded (f
) species for wild-type and
tryptophan-containing mutants can be determined; these are shown in Fig. 3, A`-D`, together with the urea
concentration (C
), at which the
intermediate is maximal. Only relatively minor differences from the
wild-type
-subunit are apparent. The midpoints, C
and C
for each transition for each protein
are given also in Table 1. Values of
G (Table 1)
obtained for the mutant proteins at 2.51 and 3.73 M (the
midpoints of the N
I and I
U transitions for wild-type
-subunit) vary between
-0.26 and 0.33 Kcal/mol. Thus, it appears that the replacement of
a phenylalanine by a tryptophan residue at either or both the 139- or
258-residue sites does not appear to significantly alter the stability
to urea.
-subunits would seem to make them ideal host proteins for the
introduction of our collection of potentially stabilizing/destabilizing
mutational alterations. Moreover, a comparison of both the UV and
fluorescence properties of the native forms of F139W and F258W and that
of the double mutant, F139W/F258W, indicates that the tryptophan
residue in each of these regions of the protein is displayed
independently spectroscopically. Therefore, the tryptophan residues
substituted at positions 139 and 258 can function as separate reporter
groups for monitoring the behavior of the putative
-1 and
-2
folding units in the intact
-subunit. The equilibrium and kinetic
unfolding and refolding properties of these mutant
-subunits
monitored by fluorescence changes, currently underway, should provide
interesting information regarding the intermediate(s) in these pathways
and also serve as the wild-type reference behavior for a study of the
effects of our other
-subunit mutants.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.