Materials and Methods
Protein expression, purification and sample preparation. A
synthetic gene encoding the HIV-1 protease monomer lacking the last 4
residues, mHIV-1-PR1-95 was a kind gift from Dr. Celia
Schiffer, University of Massachusetts Medical School, and was cloned
into a pET11a vector. The protein was expressed in Escherichia
coli Rosetta (DE3) cells upon induction with 0.2 mM IPTG. For
the synthesis of isotope labeled protein, Spectra9 LB media (Euriso-top,
France – cod. CGM-3030-CN-1, 1L) enriched with 15N
and 13C was used. Cells obtained from 0.4 L of culture
were lysed by sonication at 4º C in extraction buffer: 20mM Tris/HCl, 1
mM EDTA and 10 mM DTT, pH 8. The protein was refolded as described
previously26. For spectroscopic measurements, the
protein was dialyzed against 20 mM sodium phosphate, pH 6.0.
Fluorescence and CD experiments . Fluorescence experiments were
performed with a Varian Eclipse fluorimeter on 4 µM protein in 20 mM
sodium phosphate at pH 6.0 and 25ºC by adding different concentrations
of denaturant. CD measurements were conducted at 230 nm and a protein
concentration of 15 μM in 20 mM sodium phosphate, pH 6, and containing
different amounts of denaturant at 25°C using a JASCO J810
spectropolarimeter and a 1 mm path length. A total of 120 data points
were recorded over 1 minute and averaged. The actual urea and GdmCl
concentrations were confirmed by refractive index measurements. For the
temperature transition, CD measurements were conducted at 205 nm and a
protein concentration of 10 μM in 20 mM sodium phosphate, pH 6. The
temperature was increased in 1 °C steps from 3 to 20 °C and in 2 °C
steps from 20 to 90 °C using a Peltier control unit. To account for the
slow refolding kinetics, each point was allowed to equilibrate 5 minutes
prior to detection.
NMR experiments . Backbone assignment and
R1, R2 and hetNOE relaxation
experiments . All NMR spectra were recorded either on an Agilent DD2 800
MHz or a Varian INOVA 750 MHz spectrometer using a room temperature
probe, and standard pulse programs from the Vnmrj BioPack. For
assignment, we prepared 11 different aliquots of15N-13C-labelled
~200 μM protein solution in 20 mM sodium phosphate, pH
6.0, and 10 % D2O (v/v), 125 μM DSS
(2,2-dimethyl-2-silanepentane-5-sulfonic acid) containing 4, 6 and 8 M
urea, 0.75, 1, 2 and 4 M GdmCl, or 9% (v/v), 25% (v/v), and 45% (v/v)
acetic acid, respectively, and one containing no extra additives. For
relaxation experiments, identical samples were prepared containing a15N-labelled ~200 μM protein solution.
The backbone nuclei were assigned using HSQC35, HNCA,
HNCO36, HN(CA)CO37,
HNCOCA38, HNCACB39,
CBCACONH40, HNN41 and15N-edited NOESY-HSQC42 spectra
recorded at 25°C for the samples containing 4 and 8 M urea, and using
only HSQC, HNCA, HNCO, HNCOCA, HNN and 15N-edited
NOESY-HSQC spectra for 1 M GdmCl and 25% (v/v) acetic acid. For the
remaining samples, only the HSQC, HNCA, HNCOCA and HNCO spectra were
used for backbone assignment. The assignment was completed for 95% of
all non-proline residues for samples containing acetic acid, 96% for
samples containing GdmCl, 97% for samples containing urea and 97% for
cold denatured protein.
To analyze the T1 and T2 relaxation
times and heteronuclear NOEs (hetNOEs), five series of spectra were
recorded on 15N-labelled protein in 20 mM sodium
phosphate, pH 6.0, and 10 % D2O (v/v), 125 μM DSS, also
containing 4 or 8 M urea, 1 M GdmCl or 25% (v/v) acetic acid, at 25 ºC43. We chose 8 different delay times: 0 ms, 100 ms,
200 ms, 300 ms, 500 ms, 700 ms, 900 ms and 1200 ms for recording
T1 and 9 different delay times: 10 ms, 50 ms, 90 ms, 130
ms, 170 ms, 190 ms, 210 ms, 230 ms and 250 ms for recording
T2 relaxation times. For the hetNOE a relaxation delay
of 8 s was used.
PFG NMR diffusion experiments . The above described protein
samples were used to record sets of 60 bipolar pulse-pair stimulated
echo experiments using a watergate scheme for water suppression with
varying gradient strength 44. As internal reference,
0.5% (v/v) dioxane was added to all samples to correct for viscosity
effects by the solvent. All spectra were obtained at 25°C using 32
transients on a 750 MHz Varian INOVA spectrometer.
2-D and 3-D NMR spectra processing . The X-carrier frequency was
determined by referencing to internal DSS. The DSS frequency was
obtained from a 1D 1H spectra recorded immediately
before the remaining experiments. Indirect referencing was used in the15N and 13C dimensions by use of
conversion factors 45. The spectra were processed
using nmrPipe 46 and qMDD 47.
Spectrometer frequencies and carrier frequencies in ppm were inserted
with 4 decimals. Zero-filling to nearest power of 2 was used. The
processed spectra were assigned and analyzed in CcpNmr Analysis48. The assigned HSQC spectra were further used to
extract the relaxation decays from the series of spectra recorded to
determine the T1 and T2 relaxation times. Relaxation decay curves were
fitted to single exponentials and relaxation times determined using therelax software 49,50 The values ofR1 , R2 and the hetNOE
recorded at 17.6 Tesla were used to derive the spectral density function
at three frequencies (0, ωH and ωN)
analyzed by reduced spectral density mapping using relax49,50.
DOSY processing . Each set of 60 1D-1H spectra
was separately processed and analyzed using The DOSY Toolbox51 and MATLAB 52. Spectra were
phased in zero order and smoothed using a 10 Hz Lorentzian efficiently
removing most visible noise. The function msbackadj was used rather than
the internal DOSY Toolbox baseline correction routine.
Analysis of the chemical shifts. Secondary chemical shifts
associated with different atoms were systematized using the formula
(Δ(δCα)+Δ(δC’)-0.5*Δ(δN))7.
Fit of dynamics parameters . The R2 parameters were fitted with
the function described in Eq. 3 in the Supplementary Materials of
ref.53. The fit was done with a nonlinear least-square
algorithm employing a Levenberg-Marquardt algorithm. To avoid
overfitting, we performed fits with different number of exponentials,
eventually choosing the minimum number of exponentials which gave a
chi2 lower than 5.
NMR data have been deposited at the BioMagResBank with the accession
number: 25255.
Molecular dynamics simulations. The
mHIV-1-PR1-95 system was described with the Amber
99SBdisp force field54 in Tip4/pd water and simulated
with Gromacs 2020.455. The protein was prepared in a
dodecahedric box of 571 nm3 with 19160 water molecules
and 4 Cl- ions to neutralize the charge. A preliminary
simulation of 50 ns at 700K and constant volume was carried out,
followed by 100 ns at 300K and 1 atm. From the latter simulation, 110
conformations were extracted to act as starting conformations of the
production run. A replica-exchange simulation was then performed with
110 replicas whose temperature range from 300K to 500K for a total of 68
µs.
Once the first 30 ns were removed, the replica at 300K was analyzed to
validate the simulation against the NMR data. Secondary chemical shifts
were calculated for each conformation with Sparta+56and averaged over all of them. To calculate secondary chemical shifts,
we used Bax’s reference value56.
To predict the R1 relaxation parameters qualitatively we extracted 50
conformations from the 300K trajectory, using each of them as starting
point of a 1ns simulation at fixed temperature. The root mean square
fluctuations around each of the 50 average conformations were calculated
and then averaged together. The experimental R2 values were compared to
the solvent-accessible surface area of each residue, averaged over the
full 300K trajectory.
The clustering of the 300K trajectory was performed with a tailor-made
Python code that uses the fraction q of common contacts as
underlying metric, normalized to the maximum between the numbers of
contacts of the two structures. A contact is defined if the center of
mass of two residues are closer than 0.65 nm. In the calculation ofq, only pairs of residues which were further apart by at least 3
other residues along the chain were considered.