Results Catalytic and spectroscopic properties of tryptophan mutants of FBPase The single tryptophan mutants, Phe16Trp, Phe89Trp, Phe219Trp and Phe232Trp FBPases exhibited identical elec
Trang 1Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants
Heide C Ludwig, Fabian N Pardo*, Joel L Asenjo*, Marco A Maureira, Alejandro J Yan˜ez and Juan C Slebe
Instituto de Bioquı´mica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile
Fructose-1,6-bisphosphatase (EC 3.1.3.11, FBPase)
cat-alyzes a control step in the gluconeogenic pathway, the
hydrolysis of fructose-1,6-bisphosphate [Fru(1,6)P2] to
fructose-6-phosphate and inorganic phosphate
Diva-lent metal ions such as Mg+2, Mn+2 or Zn+2 are
required for catalytic activity [1,2] FBPase is inhibited
synergistically by AMP and fructose-2,6-bisphosphate
[Fru(2,6)P2] AMP binds to an allosteric site and its
inhibition is cooperative, whereas Fru(2,6)P2 is a
competitive inhibitor, that binds to the active site, according to structural and kinetic evidence [3,4] The pig kidney FBPase is a homotetramer having
D2 symmetry with a relative molecular mass of
146 000 [5] The crystal structures of this enzyme com-plexed with various ligands have been solved [4,6–8] (pdb: 1FPB; 1FRP; 1FBF) The four subunits of FBPase are designated C1, C2, C3 and C4 and are labeled clockwise The C1 and C2 subunits correspond
Keywords
fructose-1,6-bisphosphatase; protein
unfolding; single tryptophan mutants;
tetrameric intermediate; phase diagram
Correspondence
J C Slebe, Instituto de Bioquı´mica,
Universidad Austral de Chile, Campus Isla
Teja, Valdivia, Chile
Fax: +56 63 221406
Tel: +56 63 221797
E-mail: jslebe@uach.cl
*These authors contributed equally to this
work
(Received 18 June 2007, revised 14 August
2007, accepted 21 August 2007)
doi:10.1111/j.1742-4658.2007.06059.x
Pig kidney fructose-1,6-bisphosphatase is a homotetrameric enzyme which does not contain tryptophan In a previous report the guanidine hydrochlo-ride-induced unfolding of the enzyme has been described as a multistate process [Reyes, A M., Ludwig, H C., Yan˜ez, A J., Rodriguez, P H and Slebe, J C (2003) Biochemistry 42, 6956–6964] To monitor spectroscopi-cally the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1–C2 or the C1–C4 intersubunit interface of the tetramer The mutants were shown to retain essentially all
of the structural and kinetic properties of the enzyme isolated from pig kid-ney The enzymatic activity, intrinsic fluorescence, size-exclusion chromato-graphic profiles and 1-anilinonaphthalene-8-sulfonate binding by the mutants were studied under unfolding equilibrium conditions The unfold-ing profiles were multisteps, and formation of hydrophobic structures was detected The enzymatic activity of wild-type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum 30%) followed by a biphasic decay The activity and fluorescence results indicate that these transitions involve conforma-tional changes in the fructose-1,6-bisphosphate and AMP domains The representation of intrinsic fluorescence data as a ‘phase diagram’ reveals the existence of five intermediates, including two catalytically active inter-mediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers The intrinsic fluores-cence unfolding profiles indicate that the dimers are formed by selective disruption of the C1–C2 interface
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; FBPase, fructose-1,6-bisphosphatase; Fru(1,6)P 2 , fructose-1,6-bisphosphate; Fru(2,6)P 2 , fructose-2,6-bisphosphate; GdmCl, guanidinium chloride.
Trang 2to the upper dimer and the C3 and C4 subunits to the
lower dimer Each subunit of the enzyme can be
divided into two folding domains: residues 1–200
con-stitute the AMP domain, and residues 201–337 the
Fru(1,6)P2 domain The AMP domain has the AMP
binding site at the C1–C4 interface and the Fru(1,6)P2
domain contains the active site at the C1–C2 interface
Two quaternary conformations have been established,
the R- and the T-forms, that differ by a 17 rotation
of the lower dimer C3C4 relative to the upper dimer
C1C2 [9,10] (pdb: 1FBP; 4FBP) AMP induces the
transition from the active R-form to the inactive (or
less active) T-form
Understanding the folding⁄ unfolding and
self-assem-bly processes of oligomeric proteins remains a major
problem Equilibrium denaturation studies of such
proteins provide important information on the
rela-tionship of folding and oligomerization processes and
on the influence of quaternary structure on protein
sta-bility [11,12] In a previous publication from this
labo-ratory [13] the unfolding of pig kidney FBPase
induced by GdmCl was investigated In contrast to an
earlier study [14] that suggested that inactivation and
dissociation occur simultaneously, we demonstrated
the existence of an inactive tetrameric intermediate
Furthermore, it was shown that the equilibrium
unfolding pathway is characterized by the presence of
three intermediate states In these studies, fluorescent
reporter groups
(2-(4¢-maleimidylanilino)naphthalene-6-sulfonic acid and
N-(acetylaminoethyl)-5-naphthyl-amine-1-sulfonic acid) were chemically attached to
Cys128, a reactive thiol group located near to the
active site to monitor conformational changes and
enzyme dissociation However, the introduction of
these fluorescent groups caused a destabilization of the
active site region Furthermore, at high protein
con-centration (1 mgÆmL)1) the aggregation of dimeric and
monomeric unfolding intermediates masked the
transi-tions occurring at GdmCl concentratransi-tions above 1.2 m
However, no large aggregates have been detected by
light scattering measurements at 50 lgÆmL)1 [13]
Finally, a main unresolved question is which of the
FBPase interfaces is broken first by the GdmCl
treat-ment
As FBPase does not contain tryptophan,
introduc-tion of this fluorescent amino acid by site-directed
mutagenesis as nonperturbing probe is an attractive
experimental approach to examine the unfolding of the
enzyme at low protein concentration The tryptophan
probe, which is very sensitive to a variety of
environ-mental conditions, yields structural and dynamic
infor-mation about its surroundings [15] In the present
report, FBPase mutants carrying a single replacement
of a Phe at position 16, 89, 219 or 232 by Trp were engineered (Fig 1) Phe16 and Phe89 are residues of the AMP domain located near the C1–C4 interface, whereas Phe219 and Phe232 are in the Fru(1,6)P2 domain and near the C1–C2 interface The single-Trp mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney The GdmCl-induced unfolding transi-tions studied by fluorescence spectroscopy provide evi-dence for the existence of five unfolding intermediates and indicate that the loss of quaternary structure begins by disruption of the C1–C2 interface
Results Catalytic and spectroscopic properties of tryptophan mutants of FBPase
The single tryptophan mutants, Phe16Trp, Phe89Trp, Phe219Trp and Phe232Trp FBPases exhibited identical electrophoretic mobility ( 37 kDa) as FBPase isolated from pig kidney and were at least 96% pure using SDS⁄ PAGE as a criterion (data not shown) As seen
in Table 1, the mutations in general do not affect cata-lytic properties significantly, except the loss of AMP cooperativity (h value¼ 1) observed for Phe16Trp FBPase The other kinetic parameters only demon-strate slight differences with respect to the recombinant wild-type FBPase and are similar to those published
Fig 1 Schematic of FBPase showing the location of the trypto-phan residues Active sites and AMP binding sites are labeled FBP and AMP, respectively Dotted ovals represent ligand binding sites
on faces of the tetramer hidden from view The FBPase tetramer is
in the T-state conformation The location of the phenylalanine resi-dues which were mutated is shown.
Trang 3elsewhere for nonrecombinant FBPase [16–18] The
CD spectra of the nonrecombinant, recombinant
wild-type and mutant FBPases were essentially
superimpos-able from 200 to 250 nm (data not shown)
Emission spectra of equimolar amounts of the Trp
mutants, when excited at 295 nm, are shown in Fig 2
The emission maxima of the mutants are summarized
in Table 2 Phe16Trp and Phe219Trp FBPases have
emission maxima at 338 and 335 nm, respectively,
indi-cating that these tryptophan residues are located in a
nonpolar environment [15] In contrast, the emission
maxima of Phe89Trp and Phe232Trp FBPases are at
356 nm and 352 nm, respectively, indicating that these
tryptophan residues are in a polar environment,
exposed to the solvent Figure 2 also shows that
Phe232Trp FBPase presents the highest fluorescence
quantum yield, whereas the quantum yield of
Phe16Trp FBPase is considerably lower than those of the other mutants
The environment of a specific tryptophan residue can also be evaluated by its accessibility to a collisional fluorescence quencher, as acrylamide [19,20] Table 2 presents the results of the Stern–Volmer analysis of the quenching data of the tryptophan mutants by acrylam-ide The values of the Stern–Volmer constants (KSV) indicate that Trp219 is shielded from the solvent (KSV¼ 3.19 m)1), Trp16 (KSV¼ 5.81 m)1) and Trp89 (KSV¼ 6.28 m)1) are moderately accessible and Trp232 (KSV¼ 11.8 m)1) is almost completely solvent exposed These results agree with the crystallographic structure of the enzyme [21]
Examination of protein unfolding by catalytic activity, size-exclusion high-performance liquid chromatography and 8-anilinonaphthalene-1-sulfonate (ANS) binding
Enzyme activity can be regarded as the most sensitive probe for studying protein unfolding, as it reflects sub-tle readjustments of the active site and detects very small conformational variations of an enzyme struc-ture Figure 3 shows the changes in enzymatic activity
of the nonrecombinant, recombinant wild-type and the mutant pig kidney FBPases as a function of GdmCl
Table 1 Kinetic parameters for wild-type and single tryptophan mutants of pig kidney FBPase.
Enzyme
k cat
K m
Fru(1,6)P 2
I 50
Fru(2,6)P 2
I 50
AMP
h AMP
K a
Mg+2
Fig 2 Fluorescence emission spectra of FBPase mutants Each
enzyme was 60 lgÆmL)1in 20 m M Tris ⁄ HCl buffer, pH 7.5,
contain-ing 0.1 m M EDTA The excitation wavelength was 295 nm The
var-ious traces correspond to the following samples: – Æ –, Phe16Trp;
– – –, Phe89Trp; Æ Æ Æ Æ, Phe 219Trp; – ÆÆ – ÆÆ, Phe232 Trp; –––,
recombi-nant wild-type FBPase.
Table 2 Fluorescence properties of the single tryptophan mutants
of pig kidney FBPase The Stern–Volmer quenching constants for acrylamide (K SV ) were determined in 20 m M Tris ⁄ HCl buffer,
pH 7.5, containing 0.1 m M EDTA as described under Experimental procedures.
Enzyme
kmax nm
KSV
Trang 4concentration at 15C A similar behavior can be
observed for the six enzymes: after an enhancement in
enzymatic activity (maximum 30%) a decrease of the
activity occurs According to control experiments the
residual GdmCl concentrations (2–20 lm) in the assay
medium do not affect the enzymatic activity The
maximum activity is observed at 0.2 m (Phe89Trp,
Phe219Trp and Phe232Trp FBPases), 0.3 m (Phe16Trp
and recombinant wild-type FBPases) and 0.4 m
GdmCl (nonrecombinant FBPase) Two phases can be
distinguished in the activity decrease, an initial phase
of slight decay followed by a sharp decrease In
accor-dance with previous data [13] the midpoint for
GdmCl-based inactivation for the nonrecombinant
enzyme is 0.75 m The recombinant enzymes are less
resistant to GdmCl inactivation than the
nonrecombi-nant FBPase, as indicated by the lower denaturant
concentration required for half-maximum inactivation:
recombinant wild-type and Phe219Trp FBPases,
0.70 m GdmCl; Phe16Trp and Phe232Trp FBPases,
0.64 m GdmCl and Phe89Trp FBPase, 0.58 m GdmCl
It has been described that the inactivation of
non-recombinant FBPase takes place without dissociation
of the tetramer, and therefore the enzyme at 0.9 m
GdmCl elutes as a single peak from a size-exclusion
column pre-equilibrated with the same solvent [13]
The elution profiles of the tryptophan mutants of
FBPase at various concentrations of GdmCl were obtained (data not shown) Between 0 and 0.9 m GdmCl the enzymes elute as a single peak centered at 7.5 min, indicating that the mutants maintain their tet-rameric structure A shoulder at a higher elution time (aproximately 8.0 min) is observed in the elution pat-terns at 1.0 and 1.2 m GdmCl, indicating the presence
of dimers (relative molecular mass 70 000), as has been described for the nonrecombinant enzyme [13] ANS, a hydrophobic fluorophore, can be used as an external probe for the unfolding of proteins [22] This fluorophore has a low emission in aqueous solutions, but its fluorescence is increased in nonpolar environ-ments in such a way that the changes in ANS fluores-cence are related to the increase in accessible hydrophobic surface upon protein unfolding As shown in Fig 4, there is a sharp rise in ANS fluores-cence and thus in ANS binding to Phe89Trp FBPase between 0.4 m and 0.6 m GdmCl This transition is coincident with the loss of catalytic activity Beyond 0.7 m GdmCl the ANS emission shows a gradual decrease, reflecting the disappearance of the hydropho-bic patches where ANS binds In the case of the Phe16Trp, Phe219Trp and Phe232Trp mutants the increase in ANS binding takes place approximately between 0.5 m and 0.9 m GdmCl, and is also coinci-dent with the loss of catalytic activity These results are similar to those described for nonrecombinant pig kidney FBPase [13]
Monitoring changes of the intrinsic tryptophan fluorescence
The fluorescence of the indole ring is highly sensitive
to its environment; this makes tryptophan an ideal res-idue to detect conformational changes of protein mole-cules [15] GdmCl-induced denaturation of the tryptophan mutants was monitored by the change in fluorescence emission spectra at an excitation wave-length of 295 nm The results were plotted by taking the average emission wavelength [23] and the fluores-cence intensity at the emission maximum of each mutant in the native state versus GdmCl concentra-tion The average emission wavelength was used instead of kmax because it is a more sensitive value as
it reflects changes in the shape of the spectrum as well
as in position The unfolding curves (Fig 5) are mostly biphasic or triphasic and differ greatly in shape All of the tryptophan residues detect the transition by which enzymatic activity is lost When enzymatic activity and fluorescence were measured for the same samples of an unfolding experiment, a perfect coincidence between catalytic activity loss, change of average emission
GdmCl, M
0
20
40
60
80
100
120
140
Fig 3 Enzyme activity of wild-type and mutant FBPases, as a
func-tion of GdmCl concentrafunc-tions Samples of nonrecombinant FBPase
(d), recombinant wild-type (h), or the mutant enzymes Phe16Trp
(s), Phe89Trp (m), Phe219Trp (n) or Phe232Trp (j) (50 lgÆmL)1)
were incubated at different concentrations of GdmCl The
denatur-ant effect was then evaluated measuring enzyme activity, as
described in Experimental procedures.
Trang 5wavelength and emission intensity was obtained for
Phe16Trp and Phe89Trp FBPases
The emission intensity of Phe16Trp FBPase
increases at low GdmCl concentrations in a biphasic
way (Fig 5) The first phase, between 0 and 0.2 m
GdmCl, correlates with the increase in enzymatic
activ-ity (Fig 3), and the second phase, between 0.55 and
0.8 m GdmCl, correlates with the activity loss At
denaturant concentrations higher than 0.8 m GdmCl
the emission intensity decreases The average emission
wavelength is shifted gradually towards higher values,
and a pronounced increase of this parameter is
observed between 2.0 and 2.7 m GdmCl A probable
cause for this pronounced increase is the disruption of
the C1–C4 interface next to Trp16, which exposes the
tryptophan residue to the solvent
For Phe89Trp FBPase a large decrease of the
aver-age emission wavelength is observed between 0.4 and
0.7 m GdmCl (Fig 5) correlated with a decrease in the
fluorescence intensity Notably, the maximum emission
wavelength (kmax) value of the emission spectrum at
0.8 m GdmCl is 339 nm, characteristic for a nonpolar
environment A shift of the average emission
wave-length in the opposite direction between 1.8 and 2.5 m
GdmCl indicates that the tryptophan residue now moves into a polar environment In accordance with the results obtained for Phe16Trp FBPase, this red shift probably corresponds to the disruption of the C1–C4 interface next to Trp89
The unfolding curves of Phe219Trp FBPase, which contains a tryptophan residue located near the C1– C2 interface, have certain features differing from those of the mutants with a tryptophan residue near the C1–C4 interface The fluorescence intensity of Phe219Trp FBPase decreases in a transition that extends beyond 0.9 m GdmCl (Fig 5), a concentra-tion at which the catalytic activity is completely lost The intensity decrease and the increase of the average emission wavelength between 0.9 and 1.4 m GdmCl probably is caused by the disruption of the C1–C2 interface next to Trp219 Moreover, for Phe219Trp FBPase only a modest increase in the average emis-sion wavelength (less than 30% of the total increase) and no change of the emission intensity is detected between 1.8 and 2.7 m GdmCl The effect of GdmCl
on the fluorescence intensity of Phe232Trp FBPase is similar to that of the Phe219Trp mutant (Fig 5) This tryptophan residue, also located near the C1–C2
GdmCl, M
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Enzymatic activity, % 0 20 40 60 80 100 120 140
6 8 10 12 14 16 18 20
Phe89Trp
GdmCl, M
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Enzymatic activity, % 0 20 40 60 80 100 120 140
6 8 10 12 14 16 18
Phe232Trp
GdmCl, M
Enzymatic activity, % 0
20
40
60
80
100
120
140
6 8 10 12 14 16 18
Phe219Trp
GdmHCl, M
0
20
40
60
80
100
120
140
6 8 10 12 14 16 18
Phe16Trp
Fig 4 ANS fluorescence and catalytic activity of FBPase mutants at different concentrations of GdmCl Samples of Phe16Trp, Phe89Trp Phe219Trp and Phe232Trp FBPases (50 lgÆmL)1) were denatured by GdmCl Catalytic activity (d) and ANS emission (s) were measured as described in Experimental procedures The final ANS concentration was 40 l M
Trang 6interface, is already in a polar environment in the
native state (Table 2), and therefore only minor
changes of the average emission wavelength are
observed
Acrylamide quenching of Phe89Trp FBPase
intrinsic fluorescence
As the blue shift of the emission spectrum of Phe89Trp
FBPase during denaturation is rather unusual,
quench-ing studies were performed The Stern–Volmer plots
for acrylamide quenching are shown in Fig 6 for the
mutant in the native state and after denaturation by
different GdmCl concentrations The quenching plots
are linear within the concentration range used
Consis-tent with the changes of the average emission
wave-length (Fig 5), at 0.7 m and at 1.2 m GdmCl the
tryptophan residue is considerably more shielded
from the solvent (KSV¼ 2.90 ± 0.10 m)1 and KSV¼
2.69 ± 0.12 m)1, respectively) than in the native
state (KSV¼ 6.28 ± 0.20 m)1) At 2.4 m GdmCl,
an increase of the Stern–Volmer constant to
Acrylamide, M
F 0
0 1 2 3 4 5 6 7
Fig 6 Stern-Volmer–plots of acrylamide quenching of Phe89Trp FBPase denatured by different GdmCl concentrations Phe89Trp FBPase in 0.1 M Hepes-NaOH buffer, pH 7.5, containing 0.1 m M EDTA, 5 m M dithiothreitol and 2 m M MgSO 4 was incubated in the absence (d) or in the presence of GdmCl 0.7 M (s), 1.2 M (j) or 2.4 M (n) Quenching experiments were conducted as described in Experimental procedures The lines were obtained by fitting the data to the Stern–Volmer equation.
GdmCl, M
344
346
348
350
352
354
356
358
360
20 40 60 80 100 120 140 160
GdmCl, M
Average emission wavelength, nm 344
346
348
350
352
354
356
358
360
20 40 60 80 100 120 140 160
GdmCl, M
Average emission wavelength, nm 344
346 348 350 352 354 356 358 360
20 40 60 80 100 120 140 160
Phe16Trp
GdmCl, M
Average emission wavelength, nm 344
346 348 350 352 354 356 358 360
20 40 60 80 100 120 140 160
Phe89Trp
Fig 5 Unfolding curves of FBPase mutants monitored by tryptophan fluorescence The fluorescence emission spectra of the mutants dena-tured by GdmCl were obtained at 15 C The intensity-averaged emission wavelength (d) and the fluorescence intensity measured at the
kmaxof emission of each mutant in the native form (s) are plotted as a function of GdmCl concentration The excitation wavelength was
295 nm and the protein concentrations were: 60 lgÆmL)1 for Phe16Trp and Phe89Trp FBPases and 30 lgÆmL)1 for Phe219Trp and Phe232Trp FBPases.
Trang 78.72 ± 0.22 m)1 indicates an increased accessibility to
the solvent
Phase diagram analysis of tryptophan
fluorescence data
The method of ‘phase diagrams’ has been elaborated
by Burstein [24] for the analysis of fluorescence data
It has been shown that this method is extremely
sensi-tive for the detection of unfolding⁄ refolding
intermedi-ates of proteins [24–26] Figure 7 shows the phase
diagrams representing the unfolding of Phe16Trp
FBPase, Phe89Trp FBPase, Phe219Trp FBPase and
Phe232Trp FBPase Four independent experiments
performed with each mutant gave similar results The
phase diagram plotted for the Phe16Trp mutant
con-sists of six linear parts, corresponding to 0–0.3, 0.3–
0.5, 0.5–0.8, 0.8–1.4, 1.4–2.3 and 2.3–2.7 m GdmCl
This suggests the existence of six independent transi-tions during unfolding The intermediate that accumu-lates at 0.8 m GdmCl is the catalytically inactive tetramer, whereas the first intermediate must be a tet-rameric species of enhanced catalytic activity, as can
be deduced from Fig 3 The second intermediate that accumulates at 0.5 m GdmCl is an enzyme having approximately the same activity as the native FBPase The intermediates formed at 1.4 m GdmCl and at 2.3 m GdmCl should be dimeric and monomeric species, respectively Interestingly, the phase diagram plotted for Phe89Trp FBPase detects only one interme-diate at 0.7 m GdmCl, corresponding to the inactive tetramer Concerning these results it must be pointed out that the linearity of the parametric relationship found in a phase diagram does not necessarily indicate that the transition is of a one-step character [27] This is highlighted by the results obtained for the
Fig 7 Phase diagrams representing the unfolding of FBPase mutants induced by an increase in GdmCl concentrations The data correspond
to two independent sets of experiments performed with each mutant Denaturant concentration values are indicated in the vicinity of the corresponding symbol The fluorescence intensities of the native enzymes were taken as unity The excitation wavelength was 295 nm.
Trang 8Phe219Trp and the Phe232Trp mutants The phase
diagrams plotted for these enzymes do not detect
unfolding intermediates
Reactivation of FBPase upon dilution of GdmCl
When samples of the unfolded FBPase mutants in
3.5 m GdmCl were diluted to a concentration of 0.1 m
GdmCl the recoveries of enzymatic activity were as
follows: Phe16Trp FBPase, 60.9%; Phe89Trp FBPase,
57.2%; Phe219Trp FBPase, 59.8%; and Phe232Trp
FBPase, 63.6% These results indicate that the
unfold-ing process is not completely reversible The reduced
reversibility is similar to that observed for
nonrecombi-nant FBPase (65.8%), a value that is comparable to
previous data [13] The reduced reversibility can be
attributed to an aggregation of intermediates For this
reason the unfolding data shown in Fig 5 are only
qualitatively discussed No quantitative analysis of the
unfolding thermodynamics was attempted
Discussion
The guanidine-induced unfolding of pig kidney FBPase
has been previously studied in this laboratory using
enzyme activity, intrinsic (tyrosine) protein
fluores-cence, fluorescence of extrinsic probes and
size-exclu-sion chromatography [13] It has been shown that the
unfolding is a multistate process, involving as
interme-diates a catalytically inactive tetramer, compact dimers
and monomers As the dimeric and monomeric
inter-mediates tend to associate at the relatively high protein
concentrations (1 mgÆmL)1) used for size-exclusion
chromatography, the coexistence of aggregates with
intermediates complicates the analysis The
introduc-tion of tryptophan residues in different parts of the
protein (present work) provided us with the possibility
to further characterize the unfolding process at low
protein concentrations, detecting specific transitions
Phenylalanine and tryptophan are both neutral
non-polar aromatic amino acids, and usually substitution
of Phe for Trp does not cause large changes in the
whole protein structure As expected, the mutants
pre-sented almost the same catalytic and regulatory
prop-erties as wild-type FBPase and the CD spectra are
about the same Clearly, the structural integrity of the
enzyme was not affected The selective loss of AMP
cooperativity without loss of AMP sensitivity observed
for Phe16Trp FBPase is an effect that has been
described previously for the enzyme as a result of
chemical modification [28] or replacement by
site-direc-ted mutagenesis [16] of Lys50 The AMP cooperativity
is based on a specific signal transmission between
FBPase subunits that is lost without loss of the quater-nary structure and without loss of the cooperativity for the cofactor Mg+2, therefore it is reasonable to assume that the unfolding mechanism for Phe16Trp is the same as for wild-type FBPase
The Phe16Trp mutant has a considerably lower quantum yield than the other tryptophan mutants The local environment in protein structure can result in either very large or very small quantum yields of Trp residues [15] Examination of the three-dimensional structure of FBPase [21] reveals that the side chain of Phe16 is at distances of less than 4 A˚ from the side chains of Gln20, Asn182 and Arg198, residues that have been described as tryptophan quenchers [29,30] The quenching is partially relieved upon the first steps
of unfolding, probably because conformational changes at the tetramer level decrease the efficiency of the quenching Interestingly, the biphasic increase of fluorescence intensity correlates with the initial increase and the subsequent loss of enzymatic activity
The fluorescence equilibrium unfolding curves of the four single tryptophan mutants are very different (Fig 5) In general, changes in intrinsic tryptophan flu-orescence intensity upon protein unfolding are com-pletely unpredictable [31] The only change that can be predicted with confidence is that the spectrum will shift
to red upon greater exposure to solvent Accordingly,
we have interpreted the pronounced blue shift observed for the emission of Phe89Trp FBPase between 0.4 and 0.7 m GdmCl as the occurrence of a conformational change that causes a displacement of Trp89 into an apolar environment This kind of dis-placement is congruent with the reduced degree of exposition detected by acrylamide quenching experi-ments Concomitantly, hydrophobic patches appear on the surface of the protein, as indicated by the increase
in ANS-binding fluorescence and the catalytic activity disappears It is important to note that the four mutants remain in the tetrameric state and do not aggregate at low concentrations of GdmCl (lower than 0.9 m) as revealed by the size-exclusion experiments Furthermore, the linearity of the Stern–Volmer plots obtained for Phe89Trp FBPase by acrylamide quench-ing at 0.7 and 1.2 m GdmCl also support the idea that this mutant does not aggregate, as an aggregation should cause heterogeneity and a downward curvature
of the plots
We have interpreted the red shift near 2 m GdmCl
of the emission of Phe16Trp and Phe89Trp FBPases as
a disruption of the C1–C4 interface A similar situa-tion has been described for the Trp99Phe single trypto-phan mutant of the dimeric Trp aporepressor [23], where the emission of Trp19, a residue that is buried
Trang 9at the dimer interface, is highly red shifted upon
dis-ruption of the interface On the other hand, the
disso-ciation of the catalytically inactive tetrameric FBPase
(wild-type and mutants) into dimers begins at a
GdmCl concentration around 1 m Clearly this process
does not affect the average emission wavelength of
Trp89, which remains constant at between 0.7 and
1.8 m GdmCl, and affects only slightly the average
emission wavelength of Trp16 It can be concluded
that the interface which is disrupted first during
unfolding of FBPase is the C1–C2 interface The
results obtained with Phe219Trp FBPase are in line
with this notion
The conclusion that the C1–C2 interface is disrupted
before the C1–C4 interface might at first appear to be
at odds with the following facts: (a) In FBPase the
polypeptide chains of C1 and C2 (or C3 and C4) make
up an essential unit for catalytic activity, as they
mutu-ally exchange their Arg243 residues at the active sites
Furthermore, both chains are extensively associated
through both hydrophilic and hydrophobic
interac-tions [32]; (b) In the absence of AMP, the dimers
C1C2 and C3C4 associate primarily through
interac-tions between the side-chains of residues in two
a-heli-ces (H1 and H3) of the AMP domains When AMP
binds to the allosteric site it elicits a 17 rotation
between the dimers C1C2 and C3C4, whereas the
C1–C2 interface is essentially locked at its existing
con-formation in the R state [33] Nevertheless, the
dissocia-tion of the tetramers is preceded by the loss of
catalytic activity, and the structural changes that occur
at the active site region probably destroy some
interac-tions across the C1–C2 interface Moreover, our results
indicate that the transition by which the catalytic
activ-ity is lost not only involves conformational changes in
the Fru(1,6)P2 domain, but also at the AMP domain,
as it is detected by each of the four tryptophan
resi-dues of the mutants Therefore it is possible that this
global change causes the formation of new interactions
which stabilize the C1–C4 interface Interestingly,
Nel-son et al [34] have described a spontaneous subunit
exchange between distinct homotetramers of FBPase
to form hybrid tetramers at 4C that obviously
requires the disruption of both interfaces
The phase diagram plotted for Phe16Trp FBPase
suggests the existence of five intermediates Although
the difference in the parametric relationship between
0.3 m and 0.5 m GdmCl is moderate, it can not be
ignored, as the same change was observed consistently
in four independent experiments Then, according to
the phase diagram the first intermediate on unfolding
of Phe16Trp FBPase occurs at 0.3 m GdmCl The
exis-tence of this intermediate is also supported by the
enhancement of catalytic activity observed at low GdmCl concentrations for wild-type as well as for the mutant enzymes For the wild-type FBPase the activity enhancement has been interpreted as a local effect, caused by an increased conformational flexibility at the active site [13] Nevertheless, our present results indi-cate that the effect is not only local, as Trp16 is 30 A˚ away from the active site Unfolding intermediates at a low GdmCl concentration (around 0.1 m) have already been described for carbonic anhydrase [35] and for rabbit muscle creatine kinase [25] The phase diagram for F16W FBPase also reveals the existence of a sec-ond active intermediate at 0.5 m GdmCl The existence
of this intermediate explains the biphasic character of the inactivation of the enzymes Furthermore, this dia-gram provides the first evidence for the accumulation
of an intermediate at 1.4 m GdmCl that corresponds
to the dimer
According to the phase diagram, the Phe89Trp FBPase appears to unfold in a three-state manner (Fig 7), in which the intermediate is the inactive tetra-mer However, the existence of linearity of the para-metric relationship in a phase diagram does not necessarily indicate that the transition is of a one-step character [27] Although the transition between confor-mational states proceeds via an intermediate, the para-metric relationship can be practically linear in the following cases: (a) if the values of the measured charac-teristics of the intermediate state are close to those of the initial or final states; and (b) if the values of the measured characteristics of the intermediate state are somewhat between those of the initial and final states This is highlighted by the phase diagrams obtained for the Phe219Trp and Phe232Trp FBPases The multiple probes of the unfolding of these mutants (activity, ANS binding, tryptophan fluorescence and size-exclusion chromatography) indicate the not-one-step character of the process Nevertheless, the phase diagrams of these mutants clearly do not detect any intermediate
In conclusion, our data are consistent with the fol-lowing scheme of GdmCl-induced unfolding of FBPase:
where TN, TA1, TA2and TI are the native enzyme, the tetrameric intermediate of increased catalytic activity, the second active tetrameric intermediate and the inac-tive tetrameric intermediate, respecinac-tively; D are the dimers (C1C4 and C2C3); M and U are a monomeric intermediate and the unfolded monomer, respectively; and A corresponds to aggregates The existence of M
Trang 10is supported by the phase diagram of the Phe16Trp
mutant and by our previous report [13], in which the
fluorescence anisotropy of an
N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid-labeled FBPase and the
average emission wavelength of a
2-(4¢-maleimidylanili-no)naphthalene-6-sulfonic acid-labeled FBPase were
measured On the other hand, the inclusion of
aggre-gates in the scheme is based on previous results [13]
These aggregates are formed at high protein
concentra-tions A similar behavior has been described for rabbit
muscle creatine kinase [25] Size-exclusion
chromato-graphy studies of this enzyme show the formation of
large aggregates at a high (2 mgÆmL)1) but not at a
low (0.1 mgÆmL)1) protein concentration
Interestingly, although the unfolding behavior of
FBPase has been studied [13,14,36], the formation of
the active tetrameric intermediates TA1 and TA2 and
the notion that the loss of quaternary structure begins
by disruption of the C1–C2 interface are described
here for the first time
Experimental procedures
Materials
Fructose-1,6-bisphosphatase was purified from pig kidney
(nonrecombinant enzyme) as described previously [37]
ANS was obtained from Molecular Probes (Eugene, OR)
Auxiliary enzymes were purchased from Sigma (St Louis,
MO) and GdmCl from Merck (Darmstadt, Germany) All
other reagents were of analytical grade
Preparation, expression and purification of
FBPase mutants
Replacement of phenylalanine residues with tryptophan
was carried out using the Altered sites II in Vitro
Muta-genesis System kit, following the manufacturer’s
(Pro-mega, Madison, WI) instructions, as previously described
[16] The following mutagenic oligonucleotides were used
(the bases changed appear in bold): 5¢-GCTCACCCTAA
CCGCTGGGTCATGGAGGAGGGCAG-3¢ (Phe16Trp);
5¢-GTTAAAGTCATCTTGGGCCACCTGCGTTCTC-3¢
(Phe89Trp); 5¢-GGCTATGCCAGGGAGTGGGACCCTG
CCATCACTGAG-3¢ (Phe219Trp); 5¢-CAGAGGAAGAA
GTGGCCCCCAGA-3¢ (Phe232Trp)
The mutations were confirmed by unique restriction
enzyme digestion and by sequence analysis of the mutagenic
FBPase plasmids as described earlier [16] Protein
expres-sion and purification were performed as described [16] For
expression, the fragments encoding the wild-type or
muta-genic FBPases were excised from the corresponding plasmid
and cloned into the vector pET15b (Novagen, San Diego,
CA) The purified His-FBPases were subjected to
proteolysis with thrombin in order to remove the His-tag The protein concentration of the samples was measured using the Bio-Rad Protein assay kit with FBPase isolated from pig kidney as standard, or determined by absorbance
at 280 nm using a e1 mg⁄ mL value of 0.755 [37] for the enzyme isolated from pig kidney and 0.904 for the single-tryptophan mutants (determined by comparison with the enzyme isolated from pig kidney)
Spectrophotometric assay of fructose-1,6-bisphosphatase activity
The enzyme activity was determined spectrophotometrically
at 30C by following the rate of NADH formation at
340 nm in the presence of an excess of both glucose-6-phos-phate dehydrogenase and phosphoglucose isomerase [16,38] Unless stated otherwise, the reaction system (0.5 mL) con-tained 50 mm Tris⁄ HCl buffer, pH 7.5, 0.1 mm EDTA,
5 mm MgSO4, 30 lm Fru(1,6)P2, 0.3 mm NAD+ and 1.2 enzyme units of each auxiliary enzyme Digital absor-bance values were collected using a Hewlett Packard 8453 spectrophotometer (Hewlett Packard, Palo Alto, CA) and the linear data, from beyond the coupling lag period, were fit
to a straight line on a coupled computer using the UV-visible CHEM STATION program One unit of activity is defined
as the amount of enzyme that catalyzes the formation of
1 lmol of fructose-6-phosphate per min at 30C under the conditions described [16] Because the nonrecombinant and mutant FBPases exhibit partial substrate inhibition at high substrate concentrations, substrate saturation curves for all enzymes were fit by nonlinear regression to a modified form
of the Michaelis–Menten equation which incorporated a term for substrate inhibition [16,39] The Kavalue and the Hill coefficient (h) for Mg+2were determined by saturation curves fitting the data to the Hill equation AMP and Fru(2,6)P2inhibition curves were fit to the Taketa and Pogell equation [40] To prevent FBPase reactivation during the enzyme assay used for the examination of protein unfolding, trypsin (20 lg proteinÆmL)1) was added to the assay mixture [13]
Equilibrium unfolding
Equilibrium unfolding of FBPases was performed in 0.1 m Hepes-NaOH buffer, pH 7.5, containing 0.1 mm EDTA,
5 mm dithiothreitol, 2 mm MgSO4and GdmCl at the desired concentration The solutions were incubated for 4 h at 15C before analysis The concentration of the GdmCl stock solu-tion was determined by refractometry according to Pace [41]
Reactivation studies
The enzymes (900 lgÆmL)1) were incubated in 3.5 m GdmCl in 0.1 m Hepes⁄ NaOH buffer (pH 7.5) containing