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

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 ( T m ∼ 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 T m ∼ 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.