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An extensive thermodynamic characterization of the dimerization domain of the HIV-1 capsid protein - PubMed

An extensive thermodynamic characterization of the dimerization domain of the HIV-1 capsid protein

María C Lidón-Moya et al. Protein Sci. 2005 Sep.

Abstract

The type 1 human immunodeficiency virus presents a conical capsid formed by several hundred units of the capsid protein, CA. Homodimerization of CA occurs via its C-terminal domain, CA-C. This self-association process, which is thought to be pH-dependent, seems to constitute a key step in virus assembly. CA-C isolated in solution is able to dimerize. An extensive thermodynamic characterization of the dimeric and monomeric species of CA-C at different pHs has been carried out by using fluorescence, circular dichroism (CD), absorbance, nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and size-exclusion chromatography (SEC). Thermal and chemical denaturation allowed the determination of the thermodynamic parameters describing the unfolding of both CA-C species. Three reversible thermal transitions were observed, depending on the technique employed. The first one was protein concentration-dependent; it was observed by FTIR and NMR, and consisted of a broad transition occurring between 290 and 315 K; this transition involves dimer dissociation. The second transition (Tm approximately 325 K) was observed by ANS-binding experiments, fluorescence anisotropy, and near-UV CD; it involves partial unfolding of the monomeric species. Finally, absorbance, far-UV CD, and NMR revealed a third transition occurring at Tm approximately 333 K, which involves global unfolding of the monomeric species. Thus, dimer dissociation and monomer unfolding were not coupled. At low pH, CA-C underwent a conformational transition, leading to a species displaying ANS binding, a low CD signal, a red-shifted fluorescence spectrum, and a change in compactness. These features are characteristic of molten globule-like conformations, and they resemble the properties of the second species observed in thermal unfolding.

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Figures

Figure 1.
Figure 1.

Structure of CA-C dimer. X-ray structure of CA-C showing the dimeric structure of the domain. The monomers are depicted in different colors (gray and green). The side chains of Tyr164, Tyr169, and Trp184 are indicated as sticks in each monomer. The figure was produced with PyMOL (DeLano Scientific) and using the PDB file for CA-C (accession no. 1A43).

Figure 2.
Figure 2.

pH-induced unfolding of CA-C followed by fluorescence, ANS binding, and FTIR. Steady-state fluorescence: The 〈λ〉 (filled squares, right axis) and the maxima wavelength (open squares, left axis) are represented vs. the pH at two different CA-C concentrations. (A) 200 μM and (B) 20 μM. Experiments were acquired at 298 K. (C) ANS binding experiments, where the maxima wavelength (open squares, left axis) and the 〈λ〉 (filled squares, right axis) are represented vs. the pH. Protein concentration was 2 μM. The lines through the data are the fittings to Equation 5. Experiments were acquired at 298 K. (D) The maximum wavelength of the tyrosine band at different pHs as followed by FTIR. The line is the fit to Equation 5. Protein concentration was 1 mM. FTIR spectra were acquired at 278 K.

Figure 3.
Figure 3.

pH-induced unfolding of CA-C followed by CD and SEC. (A) Mean residue ellipticity at 222 nm at 20 μM of CA-C. (Inset) Mean residue ellipticity at 222 nm at 200 μM of CA-C. (B) Plots of the elution volume at 20 μM (open circles) and 200 μM (open squares) vs. the pH. The lines (continuous for 20 μM of CA-C; dashed for 200 μM) are the fits to Equation 5 (see text). All experiments were acquired at 298 K.

Figure 4.
Figure 4.

Thermal denaturation followed by far- and near-UV-CD, and absorbance at different pHs. (A) Selected far-UV thermal unfolding experiments at pH 4 (open squares), 6 (filled squares), and 9 (open circles) at 200 μM of CA-C. (Inset) Temperature dependence of the enthalpy change upon unfolding at 200 μM. In this temperature range, the unfolding of CA-C was characterized by a temperature-independent heat capacity change upon unfolding of 1.8 ± 0.5 kcal mol−1 K−1. The errors in the enthalpy are fitting errors to Equation 7. (B) Selected near-UV thermal unfolding experiments at pH 4 (open circles), 7 (filled squares), and 8 (blank squares) at 200 μM of CA-C. (C) The same as (B) but at 20 μM protein concentration. (D) Selected absorbance thermal unfolding traces at pH 5 (open circles), 8 (filled squares), and 9 (open squares). The scale on the Y- axis is arbitrary and thermal unfolding traces have been shifted for the sake of clarity. Protein concentrations were 200 μM. The lines are the fittings to Equation 6, taking into account that the free energy is given by Equation 7.

Figure 5.
Figure 5.

Thermal denaturation followed by protein fluorescence, ANS fluorescence, and anisotropy at different pHs. (A) Thermal unfolding traces followed by fluorescence at selected pHs for 200 μM of CA-C: pH 4 (open circles), pH 6 (white squares), and pH 9 (filled squares). (B) The thermal denaturation experiments followed by the emission intensity at 520 nm of 20 μM CA-C and 100 μM of ANS at several pHs: pH 4 (filled squares), pH 7 (open circles), and pH 11 (filled circles). The scale on the Y-axis for both fluorescence measurements is arbitrary. (C) The change in anisotropy at 20 μM (filled squares) and 200 μM of CA-C (blank squares) at pH 7. The lines are the fittings to Equation 6, taking into account that the free energy is given by Equation 7.

Figure 6.
Figure 6.

Thermal denaturation followed by FTIR. (A) The complete temperature range (280–370 K) for the FTIR experiments at pH 7, by measuring the 3/4 height width of the amide I band. Protein concentration was 1 mM. (Inset) Expansion showing the 3/4 of the height width of the band for the low-temperature transition. (B) The low-temperature transition observed by FTIR at different pHs (see label). Protein oncentration was 1 mM. (C) The low-temperature transition at different protein concentrations (see label in the figure) at pH 7. (Inset) The protein–concentration dependence of the thermal midpoint for the low-temperature transition. The errors in the temperature are fitting errors to Equation 8. The lines are the fittings to Equation 6, taking into account that the free energy is given by Equation 8.

Figure 7.
Figure 7.

The up-field shifted methyl region of the NMR spectra at pH 7 and pH 3 at different temperatures. (From top to bottom) NMR spectrum at pH 3 and 320 K; pH 7 and 320 K; pH 3 and 298 K; and pH 7 and 298 K.

Figure 8.
Figure 8.

Temperature dependence of the chemical shifts of protons in the NMR spectra. At pH 7 (see text): (A) Class I, (B) Class II, (C) Class III. At pH 3 (see text): (D) Class I, (E) Class II (methyl proton), (F) Class II (indole proton). Conditions were 200 μM of protein in phosphate buffer (pH 7) (25 mM) or deuterated acetic acid buffer (pH 3) (25 mM) with 0.1 M NaCl.

Figure 9.
Figure 9.

The GdmHCl-denaturation thermodynamical parameters at pH 7 as monitored by the change in ellipticity at 222 nm in the far-UV CD spectra. (A) Temperature dependence of the m-value from CD measurements. Errors bars are fitting errors to the LEM equation. (B) The temperature dependence of the [GdmHCl]1/2 (left axis, open squares) and ΔG (right axis, filled squares) values. The error bars are fitting errors to the LEM equation. The errors are larger at the higher temperatures, because the native baselines were shorter. The solid line represents the nonlinear least square fit of the data to formula image, which is similar to Equation 7 except that here T0, the reference temperature, was taken as 298 K. The use of this equation avoids a bias in the fitting due to the larger number of experimental data around the thermal midpoint. From this equation, the ΔHm, Tm, and ΔCp can be easily obtained. The experimental data at the Tm (right side of figure) were obtained from CD thermal denaturation experiments. ΔG values were obtained by using the mean m-value over all the temperatures. The temperature-dependence of ΔG was consistent with a temperature-independent heat capacity change, ΔCp, of 1.14 ± 0.06 kcal mol−1 K−1.

Figure 10.
Figure 10.

The equilibrium species observed in unfolding of CA-C at pH 7. U, denatured monomer; N′ partially rearranged monomer; N, folded monomer; N2, native dimer. The figures at the bottom of the scheme are for illustrative purposes only, and the rearranged reaction ongoing from N to N′ might not involve fraying of a particular α-helix. The techniques used in this work are indicated below the arrows; the temperature where the transition was observed is at the top of the arrow. The red letters indicate the transitions mapped by thermal denaturation (this work). The blue letters indicate evidence for the detection of the different transitions by chemical denaturation experiments in previous work (Mateu 2002; del Álamo et al. 2003).

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