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

Abstract

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 13 C-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.

Direct and Indirect DNP NMR Uncovers the Interplay of Surfactants with Their Mesoporous Host Material

“…38,40 The two pathways show opposite signs in the recorded NMR signal, leading to a superposition of two sets of resonances. This distinction allows for site-specific probing in crowded spectra like those obtained from protein samples 42,47 or RNA, 48 the investigation of protein−ligand binding, 43 or the determination of active dynamics under low-temperature DNP conditions. 41,49 In previous works, 50 it has been shown that DNP-enhanced ssNMR is a suitable method to probe the interactions of guest molecules with a mesoporous host materials, allowing the development of models and the description of dynamic processes inside of the pores.…”

“…The indirect polarization transfer pathway is facilitated by 1 H–X (X = 13 C, 15 N) cross-relaxation of molecular groups due to the presence of adequate dynamics for which the nuclear Overhauser effect type of mechanism is operative. , The two pathways show opposite signs in the recorded NMR signal, leading to a superposition of two sets of resonances. This distinction allows for site-specific probing in crowded spectra like those obtained from protein samples , or RNA, the investigation of protein–ligand binding, or the determination of active dynamics under low-temperature DNP conditions. , …”