Characterizing Watson–Crick versus Hoogsteen Base Pairing in a DNA–Protein Complex Using Nuclear Magnetic Resonance and Site-Specifically 13C- and 15N-Labeled DNA
2019
A(syn)-T and G(syn)-C+ Hoogsteen base pairs in protein-bound DNA duplexes can be difficult to resolve by X-ray crystallography due to ambiguous electron density and by nuclear magnetic resonance (NMR) spectroscopy due to poor chemical shift dispersion and size limitations with solution-state NMR spectroscopy. Here we describe an NMR strategy for characterizing Hoogsteen base pairs in protein–DNA complexes, which relies on site-specifically incorporating 13C- and 15N-labeled nucleotides into DNA duplexes for unambiguous resonance assignment and to improve spectral resolution. The approach was used to resolve the conformation of an A-T base pair in a crystal structure of an ∼43 kDa complex between a 34 bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (Protein Data Bank entry 1IHF), this base pair adopts an unusual Hoogsteen conformation with a distorted sugar backbone that is accommodated by a nearby nick used to aid in crystallization. The NMR chemical shifts and interp...
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
90
References
12
Citations
NaN
KQI