CONFORMATION AND STABILITY OF RECOMBINANT HIV-1 CAPSID PROTEIN P24 (RP24)

Conformation and stability of the recombinant protein HIV-1 rp24 were analyzed by circular dichroism, fluorescence spectroscopy and differen tial scanning calorimetry under different solvent conditions. From cir cular dichroism measurements, HIV-1 rp24 at pH 5.8 can be classified a s an all alpha-helical protein. A fluorescence maximum of about 330 nm indicates a predominantly hydrophobic environment of the five tryptop han residues. The GdnHCl-induced unfolding curves monitored by CD and fluorescence are sigmoidal and single phasic and the midpoints of tran sitions are independent on the protein concentration. For the calculat ion of free energy of unfolding Delta G(u)(H2O) a 'two-state' model wa s applied. The calculated values are between 18 and 24 kJ/mol and thus on the lower limit of the conformational stability of globular protei ns. Melting experiments at pH 5.8 are impaired by a strong irreversibl e aggregation at higher temperatures. However, at pH 3.0 and in the pr esence of 0.1% (w/v) octyl beta-glucopyranoside the melting curves sho w a large degree of reversibility with a T-m value of 38 degrees C and a molar enthalpy change Delta H-m of 218 kT/mol. At pH < 2.5 HIV-1 rp 24 can adopt a new conformation which is characterized by a high alpha -helical content, a strongly decreased CD in the aromatic region, a re d-shift of the fluorescence spectrum and a strong binding of ANS. Thes e spectral features of the acid-induced conformational state are simil ar to those obtained for molten globule-like folding states. HIV-1 rp2 4 unfolds cooperatively at pH 2.0 in the concentration range of about 1.5-3.0 M GdnHCl. The calculated values Delta G(u)(H2O) at pH 2.0 of a bout 12 kT/mol are significantly decreased in comparison to the Delta G(u)(H2O) values of the protein at pH 5.8.