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Protein Science (2005), 14:2387-2404. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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An extensive thermodynamic characterization of the dimerization domain of the HIV-1 capsid protein

María C. Lidón-Moya1, Francisco N. Barrera1, Marta Bueno2,3, Raúl Pérez-Jiménez4, Javier Sancho2,3, Mauricio G. Mateu5 and José L. Neira1,3

1 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain
2 Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and 3 Biocomputation and Complex Systems Physics Institute, Universidad de Zaragoza, 50009 Zaragoza, Spain
4 Departamento de Química-Física, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
5 Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, 28049 Cantoblanco Madrid, Spain

(RECEIVED December 29, 2004; FINAL REVISION May 9, 2005; ACCEPTED May 22, 2005)

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 ~ 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 ~ 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.

Keywords: circular dichroism; chemical denaturation; fluorescence; NMR; protein stability; thermal denaturation

Abbreviations: ASA, accessible surface area • {Delta}ASAtotal, the total accessible surface area exposed upon unfolding • ANS, 8-anilino-1-naphtalenesulfonate • CA, capsid protein of HIV (p24) • CA-C, C-terminal domain of CA • CA-N, N-terminal domain of CA • CD, circular dichroism • DSC, differential scanning calorimetry • FTIR, Fourier transform infrared spectroscopy • GdmHCl, guanidinium chloride • [GdmHCl]1/2, the GdmHCl concentration at the midpoint of the chemical denaturation • {Delta}Cp, the heat capacity change of unfolding • {Delta}Hm, the thermal enthalpy change at the denaturation midpoint • {Delta}S, the entropy change • HIV, human immunodeficiency virus • LEM, linear extrapolation model • NMR, nuclear magnetic resonance • SEC, size-exclusion chromatography • TSP, 3-(trimethylsilyl) propionic acid-2,2,3,3-2H4-sodium salt • Tm, the thermal denaturation midpoint • UV, ultraviolet • Ve, elution volume in SEC experiments.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041324305.

Reprint requests to: José L. Neira, Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, Avda. del Ferrocarril s/n, 03202 Elche (Alicante), Spain; e-mail: jlneira{at}umh.es; fax: +34-966658758.


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L. A. Alcaraz, M. del Alamo, F. N. Barrera, M. G. Mateu, and J. L. Neira
Flexibility in HIV-1 Assembly Subunits: Solution Structure of the Monomeric C-Terminal Domain of the Capsid Protein
Biophys. J., August 15, 2007; 93(4): 1264 - 1276.
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