Specific Signal Enhancement on an RNA‐Protein Interface by Dynamic Nuclear Polarization

Sensitivity and specificity are both crucial for the efficient solid‐state NMR structure determination of large biomolecules. We present an approach that features both advantages by site‐specific enhancement of NMR spectroscopic signals from the protein‐RNA binding site within a ribonucleoprotein (RNP) by dynamic nuclear polarization (DNP). This approach uses modern biochemical techniques for sparse isotope labeling and exploits the molecular dynamics of 13C‐labeled methyl groups exclusively present in the protein. These dynamics drive heteronuclear cross relaxation and thus allow specific hyperpolarization transfer across the biomolecular complex's interface. For the example of the L7Ae protein in complex with a 26mer guide RNA minimal construct from the box C/D complex in archaea, we demonstrate that a single methyl‐nucleotide contact is responsible for most of the polarization transfer to the RNA, and that this specific transfer can be used to boost both NMR spectral sensitivity and specificity by DNP. A guide to better spectra: Dynamic nuclear polarization, sparse isotope labeling, and methyl dynamics have been combined to yield site‐specific enhancement of NMR spectroscopic signals from the protein‐RNA binding site within a ribonucleoprotein. For L7Ae protein in complex with guide RNA from box C/D complex, a single methyl‐nucleotide contact can be spectroscopically isolated.