Article

Effects of temperature on the dynamic behaviour of the HIV-1 nucleocapsid NCp7 and its DNA complex.

Laboratoire de Génomique Structurale, CNRS UPR 9004 ESBS, Bd Sébastien Brant, Illkirch, France.
Journal of Molecular Biology (impact factor: 4). 03/2002; 316(3):611-27. DOI:10.1006/jmbi.2001.5379 pp.611-27
Source: PubMed

ABSTRACT The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers and is involved in many crucial steps of the virus life-cycle. A large number of physiological rôles of NCp7 involve its binding to single-stranded nucleic acid chains. Several solution structures of NCp7 and its complex with single-stranded RNA or DNA have been reported. We have investigated the changes in the dynamic behaviour experienced by the (12-53)NCp7 peptide upon DNA binding using (15)N heteronuclear relaxation measurements at 293 K and 308 K, and fluorescence spectroscopy. The relaxation data were interpreted using the reduced spectral density approach, which allowed the high-frequency motion, overall tumbling rates and the conformational exchange contributions to be characterized for various states of the peptide without using a specific motional model. Analysis of the temperature-dependent correlation times derived from both NMR and fluorescence data indicated a co-operative change of the molecular shape of apo (12-53)NCp7 around 303 K, leading to an increased hydrodynamic radius at higher temperatures. The binding of (12-53)NCp7 to a single-stranded d(ACGCC) pentanucleotide DNA led to a reduction of the conformational flexibility that characterized the apo peptide. Translational diffusion experiments as well as rotational correlation times indicated that the (12-53)NCp7/d(ACGCC) complex tumbles as a rigid object. The amplitudes of high-frequency motions were restrained in the complex and the occurrence of conformational exchange was displaced from the second zinc finger to the linker residue Ala30.

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Keywords

amplitudes
 
apo peptide
 
co-operative change
 
conformational exchange
 
conformational exchange contributions
 
DNA binding
 
dynamic behaviour
 
fluorescence data
 
fluorescence spectroscopy
 
increased hydrodynamic radius
 
nucleocapsid protein NCp7
 
reduced spectral density approach
 
rotational correlation times
 
second zinc finger
 
single-stranded nucleic acid chains
 
solution structures
 
specific motional model
 
temperature-dependent correlation times
 
Translational diffusion experiments
 
virus life-cycle