Denaturation of mHIV-1
Following the far-UV CD spectra of folded mHIV-1-PR1-95and cold-denatured mHIV-1-PR1-95 (Fig. S1 in the SI), we
observed nearly identical spectra over a wide range of wavelength
spanning from 208-250 nm. This is due to the presence of dominating
aromatic contributions in the far-UV region57, which
result in an atypical CD spectrum of a β-sheet protein. To monitor the
unfolding temperature of mHIV-1-PR1-95, we therefore
chose to record the mean residue ellipticity at 205 nm as a function of
increasing temperature from 3 °C to 90 °C (Fig. S2).
Besides cold denaturation occurring at 10 °C, already described in ref.27, we observed heat denaturation with an apparent
midpoint temperature, Tmapp of
approximately 50 °C and a third transition at ∼80 °C, corresponding to
the irreversible aggregation of the protein. Due to aggregation, the
heat-denatured state was not considered for high resolution NMR studies.
Under all conditions explored, the native state was never fully
populated and hence all equilibrium unfolding transitions could not be
satisfactorily fitted to a standard equilibrium transition curve.
In the presence of increasing amounts of urea,
mHIV-1-PR1-95 showed a very broad transition indicative
of a non-cooperative unfolding (Fig. 2). Interestingly, close to 2 M
urea, the unfolding transition was more than 95 % complete as judged
from CD measurements, but not according to fluorescence emission. Thus,
the data did not seem to agree with the expected behavior of a two-state
unfolding mechanism. At protein concentrations as high as those used for
the NMR experiments, mHIV-1-PR1-95 showed strong visible
aggregation making reliable measurements below 4 M urea impossible. In
all NMR experiments, the protein was > 95% unfolded as
judged from the CD signal. Monitoring the hydrodynamic radiusRh by oulsed-filed-gradient (PFG) NMR experiments
at 4 M urea showed that the hydrodynamic radius,Rh = 27.2 ± 0.5 Å, was comparable to data in ref.58. However, when increasing the urea concentration
from 4 to 8 M urea, mHIV-1-PR1-95 underwent further
expansion from 27.2 ± 0.5 Å to 28.0 ± 0.6 Å (Table 1).
Compared to urea denaturation, the equilibrium transition curve was
steeper and appeared more cooperative using GdmCl. The secondary
structure of mHIV-1-PR1-95 had already fully disappeared
in the presence of less than 0.5 M GdmCl as monitored by CD (Fig. 2).
Again, fluorescence emission indicated mHIV-1-PR1-95 to
be >95 % unfolded at a much higher concentration of
denaturant than for CD, indicating that the monomer did not follow a
two-state unfolding mechanism. At a denaturant concentration below 0.75
M GdmCl, protein aggregation was observed and NMR experiments were only
recorded when more than 95 % of the protein was denatured. Similar to
the case in urea, the Rh increased with
increasing concentration of GdmCl. For three selected samples, theRh increased from 24 ± 0.5 Å at 0.75 M GdmCl to
26.2 ± 0.5 Å at 2 M GdmCl (Table 1).
The acid denatured state appears crucial for successful refolding of the
dimeric protein59 and changes in protonation states
can result in small but distinct differences in the preferences for
local structure. The addition of just 0.1% acetic acid to 20 mM sodium
phosphate, pH 6, caused the pH of the sample to drop to 4, and in an
identical buffer containing 0.75% acetic acid, pH was 3.4. Addition of
5% acetic acid or more decreased pH below 3, where dimeric HIV-1-PR is
reported to be largely unfolded59. We observed a
midpoint of denaturation at about 0.5 % acetic acid, which corresponded
to a measured pH of 3.6. From CD experiments, further addition of acetic
acid caused additional structural changes even when full acid
denaturation was complete, when judged from fluorescence emission
spectra (Fig. 2). In addition, we observed an increase ofRh from 27.2 Å ± 0.5 at 9% acetic acid to 29.8 ±
0.6 Å at 25% acetic acid (Table 1). This increase was significantly
larger than for the other two denaturants.
Interestingly, in the absence of denaturant,
mHIV-1-PR1-95 is folded except for the N-terminal
region18. In addition, the wild-type protein folds
through a monomeric phase before dimerization24,30,60,61. Inspection of the HSQC spectrum of
mHIV-1-PR1-95 recorded in 20 mM sodium phosphate (pH
6.0) at 25 °C revealed a small but non-disputable second population.
Under these experimental conditions, the folding rate of the monomer24 is about 1 min-1, the equilibrium
thus being in the slower regime of chemical exchange for NMR
experiments. Hence, the second set of peaks most likely originated from
the denatured state D0 .