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INTRODUCTION |
An increasing number of biologically important proteins and
protein domains have been found to be only partially structured or
unstructured (unfolded) under physiological conditions (1). Notably,
many of the nuclear transcription factors show disordered transactivation domains in aqueous solution (2). It is generally accepted that the structural uniqueness of proteins determines their
biological function. Hence, the identification of unstructured proteins
raises the question: what is the structural basis of the functional
activity of such proteins/domains? Whether they act being in unfolded
state ("natively unfolded" proteins) or adopt structure upon
specific interaction with target molecules is a crucial question. The
induced-fit and acidic blob models of the function for such
transcription factor proteins represent two opposite points of view
(3). Hence, methods that allow studies of the propensity of proteins to
fold naturally are valuable.
Alcohols (trifluoroethanol, chloroethanol) have long been used to probe
the propensity of unstructured protein/domain to form secondary
structure (4-6). Their use has in part been based on the assumption
that alcohols might mimic the in vivo conditions under which
the disordered domain interacts with a target molecule. It has long
been known, however, that alcohols favor the
-helical conformation
in peptides or proteins regardless of the type of the secondary
structure the proteins/peptides form in the biologically relevant
(native) conformation (7-9). Hence, until interacting partners of
unstructured domains are identified, the current biophysical approaches
using such alcohols to drive
-helix formation present serious
difficulties in interpreting results in the context of biology.
Recently we demonstrated the extraordinary ability of a naturally
occurring solute, trimethylamine N-oxide
(TMAO),1 to force
thermodynamically unstable proteins to fold (10). Based on the two
examples studied, we have shown that TMAO can increase the population
of native state relative to denatured state by several orders of
magnitude. These proteins regained high functional activity in the
presence of TMAO. The present work addresses the question of whether
TMAO can induce an unstructured region of transcription factor for
which ordered conformation has never been identified to adopt a unique structure.
We studied large fragments of recombinant human glucocorticoid receptor
(hGR, Fig. 1), the protein that mediates the action of glucocorticoids,
hormones essential for human life. The hGR is a required intermediate
in the physiological and many of pharmacological actions of the
glucocorticoids, compounds often used for the treatment of lymphomas
and leukemias and to inhibit the immune response. The hGR has a complex
modular structure consisting of several domains: steroid-binding,
DNA-binding, and two activation function domains (AF1 and AF2), which
are acidic regions responsible for GR's post-DNA-binding
transactivation potential (11). The DNA-binding domains have defined
secondary and tertiary structure (12, 13), and by analogy with those of
other steroid receptors, so does the steroid-binding domain (14). The
isolated AF1 has been found to be unstructured in aqueous solution (4).
Here, we show that TMAO cooperatively induces structure in the hGR
1-500 fragment (GR 1-500) and that the TMAO-induced structure is
different from the
-helix-rich conformation driven by TFE. In the
smaller AF1 fragment (residues 77-262) both TMAO and TFE induced
similar structures.
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EXPERIMENTAL PROCEDURES |
Solutions of TMAO (Sigma) were prepared as described by Baskakov
and Bolen (15).
Construction and expression of GR 1-500 has been described (16, 17).
The expression vector contained a frameshift mutant of hGR coding for
amino acids 1-500 plus codons for a unique 5-amino acid sequence
followed by a stop codon. Cytosolic fractions were prepared from the
cell pellet (18). The AF1 domain fragment was extracted from hGR
cDNA digested with BglII and inserted into an expression
vector pGEX-4T-1 (Amersham Pharmacia Biotech). The recombinant
expression plasmid pGEX-4T/AF1 was selected and transformed into
Eschericha coli BL21. The bacteria containing the
recombinant vector for GST-AF1 were induced with
isopropyl-
-D-thiogalactopyranoside (0.5 mM)
for 3 h, lysed, and extracted. Insect cell cytosol containing the
expressed protein (GST-GR 1-500) or bacterial extracts containing (GST-AF1) were loaded onto a glutathione-Sepharose column at 4 °C.
After thorough washing of column, the GST-GR 1-500 or GST-AF1 bound to
the resin was incubated with alkaline phosphatase for 30 min at room
temperature to dephosphorylate the peptide. The hGR fragments were then
cleaved from GST by digesting with thrombin at 4 °C for 4 h,
collected, and concentrated using Amicon Centriprep units. Protein
purity was analyzed by SDS-polyacrylamide gel electrophoresis stained
with Coomassie Blue R-250 and estimated to be greater than 90%.
Fluorescence spectra of GR 1-500 in solution (30 µg/ml, 10 mM Tris, 10 mM NaCl, 10 mM
dithiothreitol, pH 7.9) were monitored using a Spex Fluoro Max
spectrofluorimeter (excitation 278 or 295 nm). All measurements were
made using 1-cm rectangular cuvettes thermostatted at 22 °C, and all
data were corrected for the contribution of the respective solute concentrations.
CD spectra were recorded using a Olis RSM CD Spectrometer at variable
scan rate (always slower than 60 nm/min) in 0.1-cm cuvettes thermostatted at 22 °C in 10 mM Tris, 10 mM
NaCl buffer, pH 7.9. The bandwidth was 1 nm with data spacing 0.5 nm,
and each spectrum shown is the result of 10-20 spectra accumulated,
averaged and smoothed. All spectra were corrected for the contributions
of the respective buffers.
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RESULTS |
TMAO Forces the GR 1-500 Fragment to Fold in a Cooperative
Manner--
Fig. 1 diagrams the regions
of the hGR studied, with the location of the Tyr and Trp residues
therein. Fig. 2 presents the fluorescence
emission spectra of GR 1-500 measured either upon excitation at 295 nm, to follow changes in the environment of Trp residues specifically,
or upon excitation at 278 nm, in which emission arises from Tyr and Trp
residues as well as being a result of energy transfer from Tyr to Trp
residues. Thus, the latter protocol links Tyr probes distributed
throughout the protein (Fig. 1) with fluorescence emission from the two
Trp residues. Because a substantial fraction of the fluorescence probes
of GR 1-500 is located outside of the DBD sequence (both Trp residues,
Trp213 and Trp365, and 7 out of 10 Tyr
residues, Fig. 1), the intrinsic fluorescence reflects mainly changes
involving the AF1 and adjacent regions. The quantum yield of the
emission spectra obtained in aqueous solution and in a strongly
denaturant conditions (7 M guanidine HCl) are similar,
illustrating that the extra DBD portion of GR 1-500 is largely
unfolded in this low-salt buffer solution. GR 1-500 aggregates if in
0.1 M NaCl. However, the emission spectra change
dramatically upon addition of TMAO. There is a 2.5-fold increase in
quantum yield and a shift in emission maximum from 350 nm in the
absence of TMAO to 331 nm in the presence of 3.5 M TMAO.
The fluorescence changes are typical of those accompanying the removal
of aromatic residues from polar aqueous solution into a more
hydrophobic environment. Both the increase in quantum yield and the
blue shift in fluorescence maximum indicate the formation of compact
structure in the presence of TMAO. TMAO induces the conformational
transition in GR 1-500 in a cooperative manner, as shown by monitoring
the level of fluorescence at 329 nm (upon excitation at 278 and 295 nm)
and the shift in emission maximum, as a function of TMAO concentration
(Fig. 2C).

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Fig. 1.
Diagram of human GR 1-500 with AF1 and DBD
domains highlighted. All Trp (bold vertical lines) and
Tyr (thin vertical lines) residues are shown.
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Fig. 2.
Fluorescence emission spectra (A,
295 nm excitation; B, 278 nm excitation) of GR 1-500 in the
absence of solutes (thin solid lines), in 7 M
guanidine HCl (bold solid lines), and in the presence of
1.2, 1.9, 2.8, and 3.8 M TMAO (dashed lines,
from bottom to top). C, reversible
TMAO-induced conformational transition of 1-500 fragment monitored at
329 nm emission with excitation at 278 nm (open square) or
excitation at 295 nm (open circle) or monitored by change in
emission maxima (1000/ max, filled diamond)
upon excitation at 278 nm. The nonlinear least squares best fit of
experimental data to the two-state model of protein
folding/denaturation using linear extrapolation methods (thin curve)
(31) gives apparent thermodynamic parameters of TMAO-induced folding:
G = 2.4 ± 0.9 kcal/mol, m = 1.9 ± 0.6 kcal/mol. All measurements (in A-C) were
performed in 10 mM Tris, 10 mM NaCl, 10 mM dithiothreitol, pH 7.9 at 12 °C with concentration of
protein 0.56 µM. To prevent aggregation of protein we
used proline at a constant molar ratio of TMAO:proline as 4:1 in all
samples containing TMAO.
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GR 1-500 Adopts Different Secondary Structure in TMAO and TFE
Solutions--
GR 1-500 in the absence of solute shows considerable
unfolded secondary structure in aqueous solution as measured by CD
(Fig. 3A). It is reasonable to
believe that the small amount of secondary structure observed is due to
DBD, which is known to be an independently folding domain (12, 13).
However, addition of TMAO caused significant changes in the CD spectra,
consistent with reduction of random coil conformation and formation of
secondary structure with a large contribution of
-helical structure.
The absence of an isodichroic point (the single point of intersection
of spectra) demonstrates that the conformational transition in TMAO
cannot be described in terms of a simple two-state model. The broad
peak observed at 218-222 nm in the presence of 2.37 M TMAO
may arise from the contribution of conformation other than
-helix
(perhaps
-strand). Unfortunately, we cannot estimate the
contribution of
-strand conformation in the presence of TMAO by this
method, due to high adsorption of the solvent in the far UV region. In the presence of TFE the CD spectra displays clear
-helical character with characteristic maxima at 190 nm and minima at 208 (Fig.
3B). As a first approximation, the conformational transition
of GR 1-500 in TFE can be described in terms of a "random coil" to
"
-helix" transition with the intersections of six spectra
forming an approximate isodichroic point. In contrast to the
TMAO-induced transition, which is cooperative, the TFE-induced
conformational change is noncooperative (Fig. 3C), typical
for the helix induction curves described for peptides in
TFE/H2O mixtures (19).

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Fig. 3.
A, CD spectra of GR 1-500 measured in
1.48 M TMAO (dashed line), in 2.37 M
TMAO (dotted line), and in the absence of TMAO (solid
line). B, CD spectra of GR 1-500 recorded in varied
concentrations of TFE (dashed lines, from top to
bottom at 222 nm), 10, 20, 30, 40, 50%, and in the absence
of TFE (solid line). C, the Q value
(× 10 4) at a wavelength of 222 nm plotted as a function
of TFE concentration. CD spectra were measured at protein concentration
1.02 µM (55 µg/ml) in 10 mM Tris, 10 mM NaCl, pH 7.9 at 22 °C.
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TFE and TMAO Induce Similar Conformational Change in the Isolated
AF1--
When the AF1, which lacks DBD, was expressed, both solutes,
TFE and TMAO, induce similar CD changes (Fig.
4). With this peptide, addition of either
TFE or TMAO decreases the amount of random coil character while
promoting formation of
-helical structure. The only data suggesting
that the TMAO-induced structure differs from the TFE-induced structure
is that the two solvents produce different isodichroic points (202 nm
in TFE and >205 nm in TMAO, Fig. 4). If we assume that both solutes
induce only
-helical structure in hGR AAD, the efficacy of
-helix
formation by TMAO and TFE is approximately equal (compare spectrum
obtained in 3 M TMAO with spectrum measured in 20% TFE
(2.8 M TFE)). Comparison of the differing CD spectra
obtained with GR 1-500 and the AF1 in the presence of TMAO (Fig.
3A and Fig. 4) suggests that the DBD and/or regions adjacent
to AF1 may be important for the conformational transition of AF1.

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Fig. 4.
CD spectra of hGR AF1 (amino acids 77-262)
measured in 1, 2, and 3 M TMAO (dotted
lines, from top to bottom
at 222 nm), in 20, 40, and 60% TFE (dashed
lines, from top to bottom
at 222 nm), and in the absence of solutes (solid
line). CD spectra were measured at protein
concentration 8.0 µM (160 µg/ml) in 10 mM
Tris, 10 mM NaCl, pH 7.9 at 22 °C.
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DISCUSSION |
Our fluorescence and CD data support the earlier observation that
the transactivation domain AF1 of the hGR is largely unstructured (4).
Because the hGR with AF1 deleted has only 25-30% transactivation activity of the holo-hGR, the AF1 region is clearly important for
determination of the level of transcription of genes that are under
glucocorticoid regulation (20). The AF1 of the hGR is a member of a
large family of activation domains (AAD) defined by their richness in
acidic residues and little sequence similarity (2). Since many such
domains are rich in acidic amino acids, previous work led to the idea
that acidic residues are crucial for the function of AADs (21).
According to the "acidic blob" concept (22), AADs do not adopt a
defined structure in vivo, rather it functions by general
ionic interactions with target proteins. However, mutagenesis studies
of hGR AF1 demonstrate that acidity is not the most important
determinant of activity, and negative charges per se are not
sufficient for activation (20, 23). Rather, key hydrophobic amino acids
appear to be crucial for activity (24). That the hGR AF1 might be
structured in vivo is suggested by the pattern of AF1
degradation in cell-free extracts, which show defined degradation
products that are inconsistent with the indiscriminate proteolysis
expected for an unfolded peptide (20). Structural studies have shown
that AADs have the propensity to form
-helical structure in the
presence of alcohols, and several proline substitution mutants reduce
both helix-forming potential and transactivation activity (4-6).
Keeping in mind the ability of AADs to form
-helix in the presence
of alcohols, one could hypothesize that acidic activation domains are
unstructured in vivo until they adopt a more ordered
conformation when directly involved in transcriptional activation,
according to the induced-fit model of folding (3).
The major concern over using alcohols (TFE, chloroethanol) to probe
secondary structures of peptides and proteins is the question of the
relevance of alcohol-induced structure to the biologically important
conformations of the proteins. These alcohols are such potent inducers
of
-helices that helices are forced to occur in peptides/proteins,
whereas such structures may be unlikely to exist in vivo
(7-9). As an example, the work of Hoy et al. (25) with AADs
from yeast GAL4 and GCN4 transcription factors demonstrated the ability
of AADs to adopt a
-sheet conformation under slightly acidic pH
condition, a conformation that is biologically important for
interaction with target molecules (26). TFE, on the other hand, induces
only
-helical conformation in AADs of GAL4 and GCN4 (25). This
result emphasizes the importance of the question about the
applicability of TFE in probing biologically relevant structures and
illustrates the intrinsic plasticity of the transactivation region,
i.e. the ability to adopt different conformations depending
upon solution conditions.
Our work shows that: 1) the naturally occurring solute TMAO induces
compact structure in the GR 1-500 as indicated by the substantial
enhancement in intrinsic fluorescence and blue shift of fluorescent
maxima; 2) according to the CD data this structure is different from
the
-helix-rich conformation driven by TFE; and 3) TMAO causes the
conformational transition to occur as a cooperative event, a process
characteristic of proteins with unique structures. Because
osmolyte-driven stabilization is a natural process (27), it is likely
that TMAO-induced protein folding can provide a reasonable means of
evaluating biologically relevant structures of proteins like hGR.
It has long been known that TFE increases the propensity of amino acids
to form an
-helix, presumably by strengthening the peptide hydrogen
bond in TFE/H2O mixtures and through favorable interactions
of hydrophobic amino acid side chains with TFE (19, 28). The peptide
hydrogen bonds in the helix are believed to be stabilized indirectly by
weakening the hydrogen bonding of water molecules to the peptide
backbone in the coil form (28). As a result of weakening the
hydrophobic interactions within the protein interior, TFE denatures
native proteins and promotes helical structure in most peptides and
proteins, even though this is nonnative helical structure (7-9, 29).
In contrast, TMAO increases the driving forces for protein folding. We
recently found that osmolytes use a new molecular force for protein
folding originating from the unfavorable interaction of the TMAO with
the peptide backbone (30). Due to its solvophobic effect on the
backbone, TMAO forces thermodynamically unstable proteins to fold
without altering the rules for folding to a native-like conformation
(10).
The action of both solutes, TMAO and TFE, focus on the peptide
backbone, and this ensures that the effect of both solutes are general
in scope, because the backbone is the most prevalent structural element
of the protein fabric. In opposition to TFE solution, the propensities
of hydrophobic groups to interact with solvent are essentially the same
in water as they are in TMAO solution (30). Thus, due to weakening the
hydrophobic interactions the dominant effect of TFE on protein is
denaturation with preferential formation of
-helices as a result of
strengthening peptide hydrogen bonds, whereas TMAO forces unfolded
proteins to fold by providing an additional force for folding that has
no preference for any particular secondary structure. Based on the
molecular origin of TMAO-driven protein folding, if biologically
relevant structure can be induced into the transactivation region of
hGR (or any other intrinsically unstructured AAD) without its target
molecule, it is more likely to be induced by solutes (like TMAO) that
have been selected in nature for their ability to fold and stabilize proteins than it is by alcohols.