Including Protons in Solid-State NMR Resonance Assignment and Secondary Structure Analysis: The Example of RNA Polymerase II Subunits Rpo4/7

Abstract

¹H-detected solid-state NMR experiments feasible at fast magic-angle spinning (MAS) frequencies allow accessing ¹H chemical shifts of proteins in solids, which enables their interpretation in terms of secondary structure. Here we present ¹H and ¹³C-detected NMR spectra of the RNA polymerase subunit Rpo7 in complex with unlabeled Rpo4 and use the ¹³C, ¹⁵N, and ¹H chemical-shift values deduced from them to study the secondary structure of the protein in comparison to a known crystal structure. We applied the automated resonance assignment approach FLYA including ¹H-detected solid-state NMR spectra and show its success in comparison to manual spectral assignment. Our results show that reasonably reliable secondary-structure information can be obtained from ¹H secondary chemical shifts (SCS) alone by using the sum of ¹H^α and ¹H^N SCS rather than by TALOS. The confidence, especially at the boundaries of the observed secondary structure elements, is found to increase when evaluating ¹³C chemical shifts, here either by using TALOS or in terms of ¹³C SCS.

Keywords: Rpo4/7; carbon and proton assignments; secondary chemical shifts; solid-state NMR; ssFLYA.