An extensive thermodynamic characterization of the dimerization domain of the HIV-1 capsid protein.
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.
Lidón-Moya
María C
MC
Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, Avda. del Ferrocarril s/n, 03202 Elche (Alicante), Spain.
Barrera
Francisco N
FN
Bueno
Marta
M
Pérez-Jiménez
Raúl
R
Sancho
Javier
J
Mateu
Mauricio G
MG
Neira
José L
JL
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D016880:Anisotropy; D036022:Capsid Proteins / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D002942:Circular Dichroism; D019281:Dimerization; D005453:Fluorescence; D015497:HIV-1 / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D036022:Capsid Proteins; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan