From the Department of Biophysical Chemistry,
Biocenter of the University of Basel, Klingelbergstrasse 70, CH-4056
Basel, Switzerland and § Pharmaceutical and Analytical
Development, Novartis Pharma AG, CH-4002 Basel, Switzerland
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ABSTRACT |
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The transforming growth factors- Transforming growth factors- The in vivo activation seems, however, to be more often
caused by proteolytic cleavage involving plasmin (13) or calpain (14).
A further mechanism is the enzymatic deglycosylation of the mannose
6-phosphate of the latency associated peptide (15). In addition,
protease-independent conformational changes of the latent complex
following binding to thrombospondin (16, 17) also leads to release of
active TGF- TGF- The biologically active TGF- In contrast to TGF- In this study we have characterized the solution structure of TGF- Protein Synthesis--
Biologically active, recombinant human
TGF- Sample Preparation--
The protein was provided in aqueous
solution containing 20% (v/v) isopropanol (or ethanol) and 0.1% (v/v)
acetic acid. These solutions were lyophilized, and the white, flaky
powder was stored at +4 °C. CD measurements were recorded in several
solvents and buffer systems, i.e. 10 mM HCl (pH
2.2), 5 mM Ca(H2PO4)2
(pH 4.2), 5 mM HEPES, 5 mM MES, 5 mM MOPS, 5 mM Gly-gly (6.5 < pH < 8.5), 10 mM CHES (pH 9.8), 10 mM
H3PO4, 10 mM
Na3PO4 (2.4 < pH < 11.0). TGF- UV Spectroscopy--
The UV absorption of TGF- Ultracentifugation Experiments--
A Beckman XLA analytical
ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA) equipped
with ultraviolet absorption optics was used for sedimentation velocity
and sedimentation equilibrium studies. Velocity runs were performed
with 12-mm double-sector Epon cells filled with 0.11 ml of protein
solution. The protein concentration was monitored using the UV
absorbance at 220 nm. All measurements were made with an An60 Ti rotor
316 at 20 °C with rotation speeds between 2,000 and 56,000 rpm.
Measured S values were always expressed as S20,W,
i.e. the sedimentation coefficient corrected to its value in
a solvent with the viscosity and density of water at 20 °C (30). The
molecular weight of the TGF- Circular Dichroism Spectroscopy--
CD measurements were
performed at room temperature with a JASCO J-720 spectropolarimeter,
using quartz cells with a path length of 1 cm and 1 mm, respectively.
The experimental data were expressed as mean residue ellipticity ( Spectrophotometric Titration of TGF-
The TGF- Aggregation of TGF-
The effect of several other solvents (10 mM HCl, pH 2.2, 5 mM Ca(H2PO4)2, pH 4.2)
and buffers (5 mM HEPES, 5 mM MES, 5 mM MOPS, 5 mM Gly-gly, 6.5 < pH < 8.5, 10 mM CHES, pH 9.8) on TGF- TGF-
The TGF-
The effect of NaCl on TGF- Analytical Ultracentrifugation--
The aggregation equilibrium in
the pH range of 2.3
Corresponding measurements were also performed at pH 3.9 yielding an
apparent molecular weight of 32.6 kDa, indicating a monomer (~70%)
At pH 4.1, samples were centrifuged at rotor velocities of 24,000 rpm
for 45 min. A diffuse boundary was observed containing about 75% of
the total protein. TGF-
At pH 4.3 a broad, dispersed boundary was measured that, at 5,000 rpm, yielded only one phase with a high sedimentation coefficient. TGF-
Sedimentation studies at physiological pH 7.4 failed. Even at a low
speed of 2,000 rpm no equilibrium conditions were reached. TGF-
Sedimentation studies were also performed at basic pH. Low
concentration samples of TGF- Conformational Studies of TGF-
The aggregation of the protein in the pH range 4.4 CD and UV spectroscopy as well as ultracentrifugation studies
demonstrate that TGF- TGF-
(TGF-
) are important regulatory peptides for cell growth and
differentiation with therapeutic potential for wound healing. Among the
several TGF-
isoforms TGF-
3 has a particularly low solubility at
physiological pH and easily forms aggregates. A spectroscopic
structural analysis of TGF-
3 in solution has thus been difficult. In
this study, circular dichroism spectroscopy was used to determine the
secondary structural elements of TGF-
3. In addition, the aggregation
of TGF-
3 was investigated systematically as a function of pH and
salt concentration using a rapid screening method. Sedimentation
equilibrium and sedimentation velocity analysis revealed that TGF-
3
exists predominantly in two major forms: (i) monomers in solution at
low pH and (ii) large precipitating aggregates at physiological pH.
Under acidic conditions (pH < 3.8) the protein was not
aggregated. At pH ~3.9, a monomer
dimer equilibrium could be
detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8.5. The
aggregation process was accompanied by a structural change of the
protein. The CD spectra were characterized by an isodichroic point at
209.5 nm indicating a two-state equilibrium between TGF-
3 dissolved
in solution and aggregated TGF-
3. Aggregated TGF-
3 showed a
higher
-sheet content and lower
-turn and random coil
contributions compared with monomeric TGF-
3. Both the solution structure and the aggregate structure of TGF-
3 were different from
the crystal structure. This was in contrast to TGF-
2, which showed
very similar crystal and solution structures. Under alkaline conditions
(pH > 9.8) the turbidity disappeared and a further conformational
change was induced. The pH dependence of the TGF-
3 conformation in
solution in the range of 2.3 < pH < 11.0 was reversible. Aggregation of TGF-
3 was, furthermore, influenced by the presence of
salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under
physiological conditions (pH 7.4, cNaCl = 164 mM) TGF-
3 has almost the highest tendency to aggregate
and will remain in solution only at nanomolar concentrations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
(TGF-
)1 are
multifunctional cytokines used for cellular communication. They are
called growth factors for historical reasons (1) but their main
function is to control cell proliferation and differentiation (2, 3) and to stimulate the synthesis of extracellular matrix proteins (4).
TGF-
plays a major role in the response of cells and tissues to
injury (5, 6). Five isoforms of TGF-
are known; however, only three
of them are expressed in mammalians. All isoforms show a highly
homologous sequence (>70% of conserved residues). For a given isoform
the homology between proteins from different species is >95%. The
isoforms have similar biological activities but exhibit differences in
potency depending on the target cell examined (7, 8). TGF-
is
produced by virtually all cell types as inactive precursors (1, 9) and
receptors for TGF-
are universally distributed throughout the body.
The inactive precursor is first cleaved into a latent complex, which
upon activation by acidification represents the regulation step of the
signaling process of TGF-
(7, 10). The activation of latent TGF-
in vivo by osteoclasts during bone resorption may be linked
to the acidification (pH < 3) of the osteoclast pericellular
space (11, 12).
in vivo.
2 was the first isoform for which the crystal structure could be
solved with x-ray crystallography (18-22). Next, the solution
structure of TGF-
1 was determined by heteronuclear magnetic resonance spectroscopy (23) and was found to be very similar to the
crystal structure of TGF-
2. The detailed comparison revealed only
small differences (mainly in the
-turns) which, however, could play
an important role in receptor binding and isoform recognition (24). The
crystal structure of TGF-
3 was also solved recently (25). Compared
with the TGF-
2 crystal no essential differences in the tertiary
structure were observed. Minor deviations were detected in the
N-terminal
-helix and in the
-sheet loop regions. The well
established differences in the biological activity of TGF-
2 and
TGF-
3 (25) seem to depend on differences in the surface side chains
rather than the tertiary structure. Alternatively, it could be argued
that despite the rather similar crystal structures, the two peptides
assume different structures in solution. This problem was investigated
here with CD spectroscopy.
s are homodimers consisting of two
identical chains connected via a single interchain disulfide bridge
(C77-C77), the latter being exposed to solvent. Heterodimeric TGF-
1,2 and TGF-
2,3 are also known but
rather unusual (26). Reduction of the active dimer results in the
formation of inactive monomers. Activity is not recovered by simple
reoxidation of the protein (27). The 8 other cysteine residues form 4 intrachain disulfide bridges, leading to a structural feature called
"TGF-
knot." The knot is almost inaccessible to solvent and
stabilizes the monomer structure. The monomer exhibits an elongated
nonglobular structure with dimensions of about 60 × 20 × 15 Å3. As there is only a single interchain disulfide bridge
between two monomers, hydrophobic interactions between the interface
areas are supposed to be of major importance in stabilizing the dimer (18-22). In addition, the dimer is further stabilized by a network of
hydrogen bonds, including several water molecules, located at well
defined positions in the hydrophilic cavities surrounding the
intersubunit disulfide bridge.
1 and TGF-
2, TGF-
3 shows a strong tendency
to aggregate at physiological pH, making spectroscopic measurements at
pH 7.0 rather difficult. Inspection of the crystal structure of
TGF-
3 reveals many hydrophobic residues on its surface, which could
explain its low solubility. In view of the functional differences between TGF-
2 and TGF-
3 and also of the therapeutic
potential of TGF-
3, a detailed characterization of TGF-
3
solubility and aggregation is required.
3
under a variety of conditions with CD spectroscopy. The experimental CD
spectra were deconvoluted into its secondary structural elements and
compared with the predictions derived from the TGF-
3 and TGF-
2
crystal structures. In addition, the details of the aggregation process
and the structural changes accompanying aggregation were investigated.
To this purpose, CD spectra were recorded as a function of pH, protein
concentration, and salt concentration. Analytical ultracentrifugation
measurements were performed to study the size of the aggregates while
titrating TGF-
3 from acidic to basic conditions and vice
versa. UV spectroscopy was used to monitor the turbidity of the
TGF-
3 solutions as a result of protein aggregation at different pH
and salt conditions.
MATERIALS AND METHODS
3 was prepared at Novartis Ltd. (Basel) by refolding in
vitro the monomeric, denatured protein overexpressed in
Escherichia coli(28). TGF-
3 has the sequence (24):
ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST
VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS. The proper
folding of the purified dimeric products was confirmed with reverse
phase high pressure liquid chromatography and electrospray ionization
mass spectrometry. The biological activity of the recombinant protein
was found to be identical to that of natural TGF-
3 (29). The minimum
content in TGF-
3 was about 90%, with 10% impurities due to related
substances. The molecular weight as determined by mass spectrometry was
very close to the theoretical value of 25,427 g/mol, calculated from
the TGF-
3 sequence.
3
solutions were freshly prepared 1-2 h before the measurement and
stored at +4 °C. TGF-
3 proved to be stable at room temperature for days in slightly acidic solvents. The protein concentrations investigated were in the range of 0.2 µM (5 µg/ml) to
60 µM (1.5 mg/ml) with most studies between 4 and 8 µM. TGF-
3 tends to be absorbed to the walls of the
measuring cell. This effect is significant at protein concentrations
500 nM but was negligible at the concentrations employed
in this study.
3 as a function
of pH was measured with a conventional UV spectrophotometer Specord
M500 (Zeiss, Cochem, Germany). In addition, a fast screening method
was used to study the effect of pH and NaCl on TGF-
3 aggregation.
The SPECTRAmax 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) was used as a high throughput device combining the
accuracy of a normal UV spectrophotometer with the rapidity of new
sensor technology. UV-transparent microplates (Molecular Devices) were
filled with protein solutions at various pH values between pH 2.3 and
pH 11.7 and UV spectra were recorded in the range of 250-350 nm.
Protein aggregation was monitored by measuring the turbidity at 350 nm.
The UV absorption background of the UV-transparent microplates was
subtracted from the UV absorbance of protein solutions. Special care
was taken to use exactly the same filling volume (200 µl) in each
microplate to guarantee identical path length.
3 aggregates was calculated from a
linear regression analysis of a plot of ln(optical density)
versus r2.
)
(degree·cm2/dmol). The contribution of the buffer was
subtracted. Two types of experimental settings were used for TGF-
3
solutions: the slow scan-mode was used for clear or slightly aggregated
solutions; the quick scan-mode was used for highly aggregated solutions
to prevent sedimentation during the experiment. CD spectra were
simulated by combining reference spectra of 4 different secondary
structures (
-helix,
-sheet,
-turn, and random coil). The
reference spectra were taken from Yang et al. (31) and were
derived essentially from globular proteins. The computer simulation of
the CD spectra provided a quantitative estimate of the secondary
structural elements of TGF-
3 in solution.
RESULTS
3--
Acidic (10 mM H3PO4, pH 2.3) and basic (10 mM Na3PO4, pH 11.7) stock
solutions, each containing 100 µg/ml (3.94 µM)
TGF-
3, were mixed at different ratios. The pH of the mixture was
determined, and the UV spectrum (250-350 nm) was recorded in a 1-cm
cuvette. Fig. 1A compares the
UV spectra of the two starting solutions. The absorption maximum shifts
from 277.4 nm at pH 2.3 to 284.0 nm at pH 11.7. The pH-induced changes
in UV absorption spectra were reversible, i.e. the reverse
titration from basic pH to acidic pH returned the original low pH UV
spectrum. In the pH range of 3.8
pH
10.2 TGF-
3 was
found to aggregate, causing an increase in UV absorbance due to light
scattering. This is demonstrated in Fig. 1B for pH values of
4.8, 6.3, and 10.0. The protein content in all samples was 100 µg/ml
(3.94 µM). Compared with the pH 2.3 spectrum (Fig.
1A), the shape of all spectra is distorted. Although TGF-
3 in solution does not show any absorption at
= 350 nm, the
aggregation of the peptide in the range of 4.1 < pH < 10.2 generates intensity at this wavelength due to light scattering. The
light scattering is even more pronounced at a shorter wavelength, because the light scattering intensity increases with
4.
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Fig. 1.
UV spectra of TGF- 3
as a function of pH. A, UV spectra of TGF-
3 under
acidic (100 µg/ml (3.94 µM) TGF-
3 in 10 mM H3PO4, pH 2.3) and basic
conditions (100 µg/ml (3.94 µM) TGF-
3 in 10 mM Na3PO4, pH 11.7). The maximum
absorption wavelength shifts from 277.4 nm at pH 2.3 to 284.0 nm at pH
11.7. No absorption is observed at 350 nm. B, UV spectra of
TGF-
3 (c = 3.94 µM) near physiological pH. The
protein solutions were made by mixing acidic (pH 2.3) and basic (pH
11.7) stock solutions at different ratios. Aggregation of TGF-
3 took
place at pH > 3.8 and pH < 10.2 with the aggregation
maximum around the physiological pH value. The increased absorbance at
= 350 nm is caused by light scattering and is a quantitative
measure of protein aggregation.
3 dimer contains 16 tyrosine residues. Upon ionization the
absorption maximum of Tyr shifts from
= 274.8 to 293.2 nm, with the
corresponding extinction coefficients increasing from
274.8 = 1,405 M
1
cm
1 to
293.2 = 2,381 M
1 cm
1 (32). Difference UV
spectra of TGF-
3 (3.94 µM) recorded at pH 2.3 and 11.7 yield a change in optical density at 295 nm of 0.08 for a 1 cm quartz
cell. The concentration of deprotonated tyrosine residues at pH 11.7 is
thus approximately 33.6 µM, whereas the total
concentration of tyrosine residues is 16 × 3.94 = 63 µM. The titration experiment therefore provides evidence
that only about 50% of the Tyr residues are available for
deprotonation at basic pH.
3 as a Function of pH and NaCl
Concentration--
The effect of NaCl on TGF-
3 aggregation was
studied with a microplate reader. 10 mM
H3PO4 (pH 2.3) and 10 mM
Na3PO4 (pH 11.7) stock solutions containing 64 µg/ml (2.5 µM) TGF-
3 were mixed at different ratios
in UV transparent microplates. An NaCl stock solution (400 mM) of corresponding pH was added in various increments leading to defined NaCl concentrations in the range of 36 mM
cNaCl
133 mM.
The aggregation was measured at
= 350 nm because TGF-
3 in
solution shows no absorbance at this wavelength (cf. Fig.
1A). Fig. 2 then summarizes
the absorbance at
= 350 nm measured as a function of pH and NaCl
concentration. In the absence of salt, aggregation began above pH 3.9, reached an approximate plateau between pH 4.3 and 7.1, increased again
around pH 8.8, and returned to the nonaggregated state at pH 10.8. If
the same experiment was repeated in the presence of salt, the addition of NaCl had virtually no effect in the low pH range. However, it
increased and extended the aggregation in the high pH range, e.g. in the presence of 133 mM NaCl TGF-
3 was
found to aggregate up to pH 11.6. This effect is comparable with the
"salting out" of organic (hydrophobic) molecules (33).
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Fig. 2.
Dependence of the
TGF- 3 aggregation on pH and NaCl concentration
monitored with UV spectroscopy at 350 nm. All measurements were
performed with 65 µg/ml (2.56 µM) TGF-
3 in 10 mM H3PO4, 10 mM
Na3PO4.
, cNaCl = 0 mM;
, cNaCl = 36 mM;
, cNaCl = 67 mM;
,
cNaCl = 133 mM. The absorbance at
= 350 nm is a measure of the turbidity, which, in turn, is a
quantitative measure of the aggregation process. Under acidic and very
basic conditions no turbidity increase can be observed. Aggregation
occurs in the pH range of 3.9-11.6 depending on the NaCl
concentration.
3 aggregation was also
investigated. The solubility of TGF-
3 was found to be similar in
those solvents/buffers as in phosphate buffer.
3 Aggregation As a Function of Protein
Concentration--
The aggregation of TGF-
3 was further
investigated as a function of the protein concentration in phosphate
buffer (10 mM H3PO4, 10 mM Na3PO4). At a low pH value
(<3.8) TGF-
3 could easily be dissolved up to concentrations of 150 µg/ml. No absorbance at 350 nm was observed. At approximately neutral
pH values (5.7
pH
8.6), TGF-
3 was found to strongly
aggregate. The turbidity at
350 increased linearly with
increasing protein concentration. Even at a concentration as low as 5 µg/ml (~0.2 µM), TGF-
3 precipitation was noted.
3 self-association was studied in more detail at pH 9.7, with protein concentrations between 43.3 µg/ml (1.7 µM) and 168 µg/ml (6.6 µM). The absorbance at
= 350 nm
increased with the protein concentration. A plot of the optical density versus the protein concentration yielded a straight line
intersecting the abscissa at 28 µg/ml (1.1 µM). Below
this concentration no aggregation occurred at pH 9.7. In a second type
of experiment the protein solutions (pH 9.7) were centrifuged for 30 min at 10,000 rpm in an Eppendorf centrifuge leading to a precipitation of aggregated TGF-
3. The protein concentration in the supernatant was determined with UV spectroscopy and was found to be constant with
28 µg/ml in all samples, in agreement with the turbidity measurements. At higher pH values (pH > 10.8, no NaCl), no
aggregation was observed even at high protein concentrations (data not shown).
3 aggregation at different protein
concentrations was also studied. 100 µl of an NaCl stock solution (400 mM) was added to 200 µl of TGF-
3 solution to
study TGF-
3 self-association under physiological NaCl conditions
(133 mM). Under acidic conditions (pH < 3.6) no
increase in turbidity was observed; however, at higher pH values the
addition of NaCl considerably decreased the protein solubility.
Aggregation was observed at protein concentrations as low as 3.5 µg/ml, i.e. at approximately 10 times lower concentrations
than in the absence of salt.
pH
4.2 was further analyzed with
analytical ultracentrifugation. The protein concentration was 117.1 µg/ml (4.6 µM) in 10 mM phosphate buffer.
At pH 2.3, the TGF-
3 solutions were investigated in a sedimentation
equilibrium for 16 h at 24,000 rpm. Analysis of the equilibrium
profile with a single component model yielded a molecular mass of 26.1 kDa, which is in agreement with the theoretical molecular weight of the
TGF-
3 monomer (25.4 kDa).
dimer (~30%) equilibrium.
3 was found to associate in large nonspecific
aggregates. Next, the samples were centrifuged for 3 h at 56,000 rpm. A second boundary was observed representing about 25% of the
total protein and corresponding to the monomeric protein. At pH 4.1, TGF-
3 thus exists in two forms only, namely monomers (~25%) and
large aggregates (~75%).
3 was completely aggregated and formed large nonspecific aggregates.
3 was
found to associate into large aggregates.
3 (40 µg/ml) at pH 9.7 were
centrifuged at 10,000 rpm, measuring the change in protein
concentration at the limit of the UV detection system. Even under this
low concentration conditions no sedimentation equilibrium was reached
with the mass of TGF-
3 aggregates being still too large to be
measured. This result is consistent with the turbidity measurements
discussed above (Fig. 2) indicating TGF-
3 aggregation at protein
concentrations
30 µg/ml at pH 9.7. The results obtained with
analytical ultracentrifugation are summarized in Table
I.
Sedimentation measurements of TGF-3
3 concentration, 120 µg/ml; T, 20 °C; buffer, 10 mM H3PO4, 10 mM
Na3PO4
3--
The variation of the UV
spectra with pH (Fig. 1A) suggests a conformational change
of TGF-
3. To obtain insight into the molecular details of this
process, CD spectra were measured in the pH range of 3.2
pH
4.4 and at pH 9.8 and are shown in the Fig.
3. In the intermediate pH range, the
protein solutions were turbid and allowed only an approximate
interpretation of the spectra. Inspection of Fig. 3 reveals an
isodichroic point at
= 209.5 nm, providing evidence for a two-state
conformational equilibrium at low pH. The pH-induced conformational
change was found to be reversible. The CD spectra at acidic pH are
characteristic of essentially a
-sheet structure with additional
contributions from random coil,
-turn, and
-helix (see below).
The spectra at very basic pH are characterized by a distinct minimum
around 217 nm and a positive ellipticity below 200 nm, indicative of an
increased contribution of
-sheet conformation at high pH values.
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Fig. 3.
CD spectra of TGF- 3
as a function of pH in 10 mM H3PO4,
10 mM Na3PO4 mixtures.
, pH
3.2;
, pH 4.0;
, pH 4.4;
, pH 9.8. An isodichroic point is
observed at
= 209.5 nm.
pH
9.8 leads to CD spectra that are distorted by light scattering. However, the observation of an isodichroic point provides evidence that
the CD spectra, as far as measurable, have a shape intermediate between
those shown in Fig. 3 for pH 4.4 and pH 9.8.
DISCUSSION
3 is soluble in monomeric form at pH
3.8 and pH
9.7. In contrast, the peptide aggregates at
intermediate pH values with the aggregation maximum occurring at
6.8
pH
8.2 (cf. Fig. 2). Based on the amino
acid sequence (24) and on the known pK values of amino acids
free in solution (neglecting pK shifts induced by intrachain
interactions), it is possible to calculate the net charge of TGF-
3
as a function of pH using the Henderson-Hasselbach equation. An
isoelectric point is estimated for pH = pI
6.8. The
solubility of TGF-
3 at low and high pH values could thus be
explained by its large positive or negative electric charge,
respectively, at these pH extremes. In contrast, aggregation at
physiological pH could be induced by the rather hydrophobic surface of
the electrically neutral protein. Analogous calculations for TGF-
1
and TGF-
2 yield isoelectric points of pI ~ 9.5 and pI ~ 8.5, respectively. In fact, TGF-
1 and TGF-
2 are distinctly
more soluble than TGF-
3 under physiological conditions. The good
solubility of TGF-
3 at low pH could explain its prominent role
in vivo in processes involving acidification of the
surroundings. The most striking examples are: (i) bone remodeling with
pH < 3.0 around the osteoclasts (34-37); (ii) inflammation where
lysosomal release can locally lower the pH under 5.0 (38-40); and
(iii) the ubiquitous expression of TGF-
3 in gastric tissues and its
strong influence in gastric cancers, in contrast to TGF-
1, which is localized principally in parietal cells, and TGF-
2, which is present
exclusively in chief cells (41).
2 was the first TGF-
isoform to be crystallized (at pH 4.5).
The x-ray analysis yielded a structure with 11%
-helix, 36%
-sheet, 8% helical turns, 3% 310-helical turn and 42%
random coil (22). In 1996, the crystal structure of TGF-
3 was also solved (25). Comparison with TGF-
2 revealed, however, only small
differences, mainly in the
-loop regions (25). Because the
percentages of structural elements were not specified by Mittl et
al. (1996), TGF-
2 was taken as the starting point for the simulation of the CD spectra. Based on reference CD spectra of 18 globular proteins (31) and using the percentages of the crystal structure, the CD spectrum shown by the dotted line in Fig.
4 was calculated. The theoretical
spectrum is quite different from the experimental spectrum (solid
line), indicating that the TGF-
2/TGF-
3 crystal structure is
not a good model for TGF-
3 in solution. A much better fit to the
experimental spectrum at pH 4.4 is given by a simulation containing 4%
-helix, 66%
-sheet, 8%
-turns, and 22% random coil (
in
Fig. 4). Compared with the crystal structure, the
-helical content
is reduced and the contribution of
-structures is clearly enhanced.
The displacement in the wavelength of the two spectra is due to
spectral distortions caused by light scattering of the TGF-
3
aggregates.
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Fig. 4.
Comparison of experimental and simulated CD
spectra of TGF- 3. Dotted line,
simulated CD spectrum based on the TGF-
2/-
3 crystal structure,
pH ~ 4.5; solid line, experimental CD spectrum of
TGF-
3 in 10 mM H3PO4, 10 mM Na3PO4, pH 4.4. The experimental
spectrum is distorted and shifted due to light scattering and optical
flattening of the slightly turbid solution.
, simulation of the
TGF-
3 CD spectrum with a secondary structure of 4%
-helix, 66%
-sheet, 8%
-turns, and 22% random coil.
The simulation of CD spectra is a multiparameter fit, and the relevance
of the structural parameters is often subject to criticism. However, CD
simulations are biased in sensitivity toward -helical structures.
The decrease of
-helix of TGF-
3 in solution compared with the
crystal structure is unambiguous and clearly beyond the error of the
numerical approach. A possible explanation for the reduced
-helical
content of TGF-
3 in solution is the presence of glycine (Gly-63) in
the
-helical region of TGF-
3 between amino acids 57 and 68. This
glycine, which confers to the protein backbone additional flexibility,
is not present in the
-helical regions of TGF-
1 and TGF-
2
(25).
CD spectra of TGF-3 in solution at pH 1.9 have been reported
previously, indicating a much larger helix content (28). We have not
been able to confirm these results, which were probably caused by
excessive smoothing of the spectra. The present findings are, however,
in agreement with 2D-NMR studies of TGF-
3, which suggest an
increased molecular flexibility in comparison with TGF-
1.2 The considerable
structural difference between TGF-
3 in solution and the crystal
structure suggests a rather flexible conformation that can adjust
itself to external constraints. It could explain the different receptor
specificity of TGF-
2 and TGF-
3 despite similar x-ray structures.
We have also recorded CD spectra of TGF-2 in solution at pH 2.96 (data not shown) and have compared them with theoretical spectra
calculated on the basis of the crystal structure. In contrast to
TGF-
3, a good agreement between the experimental and theoretical spectral shapes was found for TGF-
2. The CD results for TGF-
2 may
serve as a positive control for the sensitivity of CD spectroscopy to
detect conformational changes for the problem at hand. They emphasize
that TGF-
2 adopts the same structure in the crystal and in solution,
whereas TGF-
3 reveals two different conformations.
The CD spectra of TGF-3 further demonstrate that the conformation of
this protein varies with the pH of the solution. A first conformational
change occurs at pH ~ 4.4 and is accompanied by TGF-
3
aggregation; the second transition begins at pH ~ 9.8 and is
associated with the deprotonation of the solvent-accessible tyrosine
residues (8 of 16; see above). At the same time the aggregation process
is reversed. The pH-induced aggregation can also be detected with
fluorescence spectroscopy using the TGF-
3 Trp residues as intrinsic
markers. Aggregation leads to an increase in the steady state
polarization and an increase of the fluorescence life
time.3 A deconvolution of the
CD spectra was attempted in the pH ranges of 2.2
pH
6.0 and 9
pH
10.4 where aggregation was not too pronounced.
The relative contributions of the different secondary structures are
summarized in Fig. 5. The most prominent
change is the increase in
-structure around pH 4.4, which is
reversed at pH ~ 9.8.The destabilization of the
-structure at
pH ~ 9.8 is compensated by an increase in
-helix.
|
Concluding Remarks--
In conclusion, the solubility of TGF-3
under physiological conditions (pH 7.0) is low and distinctly smaller
than that of TGF-
1 and TGF-
2. TGF-
3 has a high tendency to
adsorb to hydrophobic surfaces and to form large aggregates. At extreme
pH values (pH < 2.3 or pH > 11.3) TGF-
3 is monomeric in
solution, whereas at pH ~ 3.9 a monomer
dimer
equilibrium was detected by ultracentrifugation. The addition of salt
greatly reduces the solubility of TGF-
3 and enhances its tendency to
aggregate. The structure of TGF-
3 in solution is different from the
crystal structure; notably, the helix content is reduced, and the
-structure content is increased. The change in conformation of
TGF-
3 in solution could explain the different receptor specificity
of TGF-
3 compared with TGF-
2 despite their very similar x-ray structure.
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ACKNOWLEDGEMENT |
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We thank A. Lustig, Biocenter of the University of Basel, for the analytical ultracentrifugation experiments.
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FOOTNOTES |
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* This work was supported by the Swiss National Science Foundation Grant 3100-42058.94.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 41-16-267-2191; Fax: 41-61-267-2189; E-mail: seelig1{at}ubaclu.unibas.ch.
2 M. J. Blommers and T. Arvinte, unpublished results.
3 T. Arvinte, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
TGF-, transforming growth factor-
;
MES, 4-morpholineethanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid;
CHES, 2-(cyclohexylamino)ethanesulfonic acid.
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REFERENCES |
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