Sensing peptide-oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein.
We present a new methodology for site-specific sensing of peptide-oligonucleotide (ODN) interactions using a solvatochromic fluorescent label based on 3-hydroxychromone (3HC). This label was covalently attached to the N-terminus of a peptide corresponding to the zinc finger domain of the HIV-1 nucleocapsid protein (NC). On interaction with target ODNs, the labeled peptide shows strong changes in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from the bulk water by the ODN bases. Remarkably, this two-color response depends on the ODN sequence and correlates with the 3D structure of the corresponding complexes, suggesting that the 3HC label monitors the peptide-ODN interactions site-specifically. By measuring the two-color ratio, we were also able to determine the peptide-ODN-binding parameters and distinguish multiple binding sites in ODNs, which is rather difficult using other fluorescence methods. Moreover, this method was found to be more sensitive than the commonly used steady-state fluorescence anisotropy, especially in the case of small ODNs. The described methodology could become a new universal tool for investigating peptide-ODN interactions.
Shvadchak
Volodymyr V
VV
Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 du CNRS, Université de Strasbourg, 67401 Illkirch, France.
Klymchenko
Andrey S
AS
de Rocquigny
Hugues
H
Mély
Yves
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D002867:Chromones / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D005663:Furans / Q000737:chemistry*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000378:metabolism
D002867:Chromones; D005456:Fluorescent Dyes; D005663:Furans; C071884:NCP7 protein, Human immunodeficiency virus 1; D009841:Oligonucleotides; D010455:Peptides; D054301:gag Gene Products, Human Immunodeficiency Virus