Cu(II)-Based Paramagnetic Probe to Study RNA-Protein Interactions by NMR.
Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein's binding site using "click" chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5'-ACGCU-3'. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagnetic probe as the result of RNA-protein interactions. The extent of the attenuation was related to the probe's proximity allowing us to construct the protein's contact surface map.
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Leah M
LM
Seebald
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Christopher M
CM
DeMott
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Srivathsan
S
Ranganathan
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Papa Nii
PN
Asare Okai
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Anastasia
A
Glazunova
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Alan
http://orcid.org/0000-0001-8246-2935
A
Chen
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Alexander
A
Shekhtman
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Maksim
http://orcid.org/0000-0002-2930-2171
M
Royzen
D066241:Carbon-13 Magnetic Resonance Spectroscopy; D057930:Click Chemistry; D056831:Coordination Complexes / Q000138:chemical synthesis / Q000737:chemistry*; D003300:Copper / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D066244:Proton Magnetic Resonance Spectroscopy; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
D056831:Coordination Complexes; C071884:NCP7 protein, Human immunodeficiency virus 1; D009843:Oligoribonucleotides; D054301:gag Gene Products, Human Immunodeficiency Virus; D003300:Copper