Download Site-selective probing of cTAR destabilization

Site-selective probing of cTAR destabilization highlights the
necessary plasticity of HIV-1 nucleocapsid protein to chaperone the
first stand transfer.
Julien Godet1,2, Cyril Kenfack1,3, Frédéric Przybilla1, Ludovic Richert1, Guy Duportail1, Yves Mély1,*
Supplementary
Time-resolved experiments
In time-resolved anisotropy measurements, the emission was collected at directions parallel I//(t) and
perpendicular I(t), alternatively, to the excitation beam polarization (interposition of a quartz crystal rotating the
beam polarization by 90°). Time-resolved anisotropy decays were analysed according to:
I// = I(t)[1 + 2 r(t)]/3
Equation (S1)
I┴ = I(t)[1 - r(t)]/3
Equation (S2)
r (t ) 

I //  G  I 
 r0  i  exp  t
i
I //  2G  I 
i

Equation (S3)
where G is a correcting factor at the wavelength λ of emission (G values were 1-1.02). The shorter component
1, associated to the internal motion of the probe, could be modelled as due to hindered rotation (75).
The theoretical values of the rotational correlation times were calculated from the molecular mass (M) of the
complexes, assuming a spherical shape, by M ( + h) / RT, where  is the viscosity (assumed to be 1 cP),
T is the temperature (293K),  is the specific volume of the species (assumed to be 0.83 mL/g (56)), h is the
hydration degree (assumed to be 0.2 mL/g for proteins) and R is the molar gas constant.
The cone semi-angle (0) of the angular range of the hindered rotation is given by
0  cos 1 (0.5  ( 1  8S  1))
Equation (S4)
0.5
  '   '3 
 where ’2 and ’3 are the amplitudes
where S is the generalized order parameter defined by S   2
 r0

of the rotational correlation times, recalculated such that ’i = r0.
Validation of the determination of the shortest lifetime and rotational correlation times was previously described
elsewhere 31.
Titration experiments:
Reverse titrations were performed by adding increasing concentrations of cTAR Lai to a fixed amount of NC(1155) (Figure 3). The binding was monitored via the quenching of the Trp37 fluorescence induced by its stacking
with the oligonucleotide bases (33, 35, 36). Binding experiments were performed in a buffer containing 25 mM
of TRIS, 30 mM NaCl, 0.2 mM of MgCl2, pH 7.5. Fluorescence intensities were corrected for dilution, buffer
fluorescence and screening effect due to the absorption of nucleic acids. Affinity constants were determined from
direct fitting of the experimental data corrected for dilution and screening effect according to the rewritten
Scatchard equation
I  Ii 
( I p  I i )  1  K  1  nx NC tot  

n


1  K 1  nxNC 
2

2 K  NC tot
tot
2
2
 4nxK  NC tot 



Equation (S5)
where I is the fluorescence intensity at a given cTAR concentration, Ii and Ip are the fluorescence emission
intensities at 320 nm (where the 2Ap does not fluoresce) in the absence of oligonucleotide and at the plateau
value respectively. [NC]tot is the total concentration of NC(11-55), Kµ is the apparent dissociation constant, x is
the ratio [cTAR]tot/[NC]tot. The number of binding sites n is ascertained from the intercept of the initial slope of
the titration curve with the asymptote to the final plateau. The parameters are recovered from non-linear fits of
Equation (S5) to experimental datasets with a home-written Igor Pro 6® (WaveMetrics) fitting procedure.
The number of peptides bound per cTAR was 10.7+/-0.3 for NC(11-55) (Figure S1 inset) giving an occluded
binding site of about 5 nucleotides per peptide, in agreement with the previously reported binding size of HIV-1
NC on nucleic acids (5,34,36). No significant differences were induced by the substitution of an adenine by a 2Ap in the cTAR sequence (for any 2-Ap insertion), indicating that 2-Ap does not affect NCp7 binding (Figure
S1). The apparent binding constant Kµ can be retrieved from the experimental data, using a model of n identical
and independent binding sites for NC(11-55), according to Equation (S5) (32). Although it is obvious that the
latter assumption is approximate (NCp7 binding depends on the oligonucleotide sequence and strandedness), this
approach yields a Kµ value of 1.4×107 M-1, which is in good agreement with previous reports on cTAR Mal
strain (28,32).
Figure S1: Temperature dependence of cTAR 2-Ap35.
When the 2-Ap is located in a single-stranded segment (internal loop), no melting transition can be observed
when the temperature increases.
Figure S2: Representative time-resolved anisotropy decay of cTAR labelled by 2Ap at position 9 in the presence
of increasing amounts of NC(11-55). Continuous lines represent the fit of the data according to equation S3. The
decays are recorded in the absence (red) or in the presence of 1 (green), 4 (blue), 7 (cyan), 10 (purple) and 15
(yellow) equivalents of NC(11-55).
Figure S3: Lifetime distribution of cTAR labelled by 2-Ap at position 53 obtained by the maximum entropy
method in the absence (hashed) or in the presence of NC(11-55) (grey).
Figure S4: Binding curve of NC(11-55) to cTAR Lai.
Substituting the natural A by a 2-Ap did not affect the binding of cTAR to NC(11-55), as seen from the perfect
superimposition of the titration curves of the unlabelled and labelled cTAR (only AP45 and AP28 are represented
together with the unlabelled cTAR for clarity). The solid line corresponds to the fit of the data with equation S5.
Binding experiments were performed in a buffer containing 25 mM of TRIS, 30 mM NaCl, 0.2 mM of MgCl2,
pH 7.5.The concentration of NC(11-55) was 1µM. Inset: The binding stoichiometry of NC(11-55)/cTAR
complex was inferred from the intercept of the initial slope with the plateau of the titration curve.
Figure S5: Evolution of the 2-Ap quantum yields in response to increasing NC(11-55) concentrations.
Bars give the QY of 2-Ap-labeled cTAR species in the presence of 1, 4, 7, 10 and 15 equivalents of NC(11-55)
(from dark to light grey). Error bars corresponds to SD for at least 3 independent experiments. The QY of the 2Ap residues were increased, although differently, by NC(11-55) in almost all the 2-Ap positions, showing that
NC(11-55) binds all over the cTAR sequence.
Figure S1.
Figure S2.
Figure S3.
Figure S4.
Figure S5.