Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine.
The nucleocapsid protein (NC) plays an important role in HIV-1, mainly through interactions with the genomic RNA and its DNA copies. Though the structures of several complexes of NC with oligonucleotides (ODNs) are known, detailed information on the ODN dynamics in the complexes is missing. To address this, we investigated the steady state and time-resolved fluorescence properties of 2-aminopurine (2Ap), a fluorescent adenine analog introduced at positions 2 and 5 of AACGCC and AATGCC sequences. In the absence of NC, 2Ap fluorescence was strongly quenched in the flexible ODNs, mainly through picosecond to nanosecond dynamic quenching by its neighboring bases. NC strongly restricted the ODN flexibility and 2Ap local mobility, impeding the collisions of 2Ap with its neighbors and thus, reducing its dynamic quenching. Phe(16)-->Ala and Trp(37)-->Leu mutations largely decreased the ability of NC to affect the local dynamics of 2Ap at positions 2 and 5, respectively, while a fingerless NC was totally ineffective. The restriction of 2Ap local mobility was thus associated with the NC hydrophobic platform at the top of the folded fingers. Since this platform supports the NC chaperone properties, the restriction of the local mobility of the bases is likely a mechanistic component of these properties.
Avilov
S V
SV
Institut Gilbert-Laustriat, UMR 7175 CNRS/Université Louis Pasteur (Strasbourg I), Dépt. Pharmacologie et Physicochimie, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France.
Piemont
E
E
Shvadchak
V
V
de Rocquigny
H
H
Mély
Y
Y
D015075:2-Aminopurine / Q000737:chemistry*; D000595:Amino Acid Sequence; D005456:Fluorescent Dyes / Q000737:chemistry*; D015497:HIV-1 / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics
D005456:Fluorescent Dyes; C071884:NCP7 protein, Human immunodeficiency virus 1; D009841:Oligonucleotides; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus; D015075:2-Aminopurine