Direct detection of N-H[...]N hydrogen bonds in biomolecules by NMR spectroscopy.
A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]N hydrogen bonds (H-bonds) in 15N isotope-labeled biomolecules. This quantitative HNN-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect magnetically active (15)N nuclei of the H-bond donor and acceptor. Detectable H-bonds comprise the imino H-bonds in canonical Watson-Crick base pairs, many H-bonds in unusual nucleic acid base pairs and H-bonds between protein backbone or side-chain N-H donor and N acceptor moieties. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor in a single experiment. The size of the scalar couplings can be related to H-bond geometries and as a time average to H-bond dynamics. The time required to detect the H-bonds is typically less than 1 d at millimolar concentrations for samples of molecular weight < or = approximately 25 kDa. A C15N/13C-labeled potato spindle tuber viroid T1 RNA domain is used as an example to illustrate this procedure.
Dingley
Andrew J
AJ
Department of Chemistry and School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
Nisius
Lydia
L
Cordier
Florence
F
Grzesiek
Stephan
S
D002247:Carbon Isotopes; D006860:Hydrogen Bonding; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D012367:RNA, Viral