Analytical Sciences

Analysis of Binding Mode of 2′-GMP to Proteins Using ¹H/³¹P NMR Spectroscopy

Noriyuki SUKA,* Kaori OKIZUMI,* Kazuo FURIHATA,** and Mitsuru TASHIRO*

*Department of Chemistry, College of Science and Technology, Meisei University, Hino, Tokyo 191-8506, Japan
**Division of Agriculture and Agricultural Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan

¹H/³¹P NMR techniques were applied to analyze the binding mode of guanosine 2′-monophosphate (2′-GMP) to histone. To date, no structures of the complex comprising 2′-GMP and histone have been deposited in Protein Data Bank. Because the ³¹P nucleus can be a selective marker of phosphorylated compounds, the combined use of ¹H and ³¹P NMR spectroscopy has been applied to investigate these molecular interactions. The complex formation was initially confirmed by ³¹P-diffusion ordered spectroscopy and ³¹P-T₁ measurements. In ¹H{¹H} saturation transfer difference experiments, H2′ and H3′ signals of 2′-GMP were significantly attenuated, while the rest of the unexchangeable protons were observed, indicating that the contribution of H2′ and H3′ to the binding epitopes was low. The WaterLOGSY-type experiment with ³¹P detection also indicated that a phosphorylated group located close to H2′ and H3′ had little access to histone.

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