Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

Abstract

Solid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.

Paramagnetic Solid‐State NMR to Localize the Metal‐Ion Cofactor in an Oligomeric DnaB Helicase

“…[32] b. Average PCS calculated over the assigned 15 N, 13 C α and 13 C β PCS extracted from 2D NCA and 3D NCACB spectra. All residues which are not visible in the paramagnetic spectrum are highlighted by light red bars.…”

“…Since ATP‐binding is accompanied by the binding of Mg 2+ , [6] substitution of Mg 2+ by paramagnetic ions, such as Mn 2+ or Co 2+ , allows us to introduce paramagnetic probes into the protein assembly. This approach is commonly applied in NMR as well as EPR and paramagnetic effects are often used in structure calculation protocols [7–16] . The oligomeric character of DnaB and thus its inherent size, limit however the use of solution‐state NMR due to the broadening of the lines induced by fast T 2 relaxation for slowly tumbling large proteins (life‐time broadening effects) [17,18] .…”

“…This approach is commonly applied in NMR as well as EPR and paramagnetic effects are often used in structure calculation protocols. [7][8][9][10][11][12][13][14][15][16] The oligomeric character of DnaB and thus its inherent size, limit however the use of solution-state NMR due to the broadening of the lines induced by fast T 2 relaxation for slowly tumbling large proteins (life-time broadening effects). [17,18] Solid-state NMR is not affected by this size limitation and offers also significant advantages compared to X-ray crystallography in that it is wellsuited for the investigation of difficult-(or even impossible)-tocrystallize biomolecular assemblies.…”

“…PRE effects can be observed for both, Mn 2 + and Co 2 + , although they are expected to be larger for Mn 2 + due to the shorter T 1e relaxation times of Co 2 + compared with Mn 2 + . [9,19,[33][34][35] An example using Co 2 + as a paramagnetic center at a static magnetic field of 20 T showed that all 13 C nuclei with a distance of less than 10 Å to the paramagnetic metal ion are broadened beyond detection. [35] Residues in the vicinity of the Co 2 + metal center experience additional large anisotropic paramagnetic shifts (resulting from the anisotropic part of the hyperfine interaction [36] ) precluding their detection in solid-state NMR spectra at high magnetic fields and slow-to-moderate MAS frequencies.…”

Protein resonance assignment by solid-state NMR based on 1H-detected 13C-based double-quantum spectroscopy at fast MAS

“…Solid-state nuclear magnetic resonance spectroscopy (SSNMR) is a powerful technique to study insoluble, aggregated or non-crystalline biomaterials, ranging from biopolymers ((Kelly et al 2020), (Zhao et al 2020),(Goldberga et al 2018)), carbohydrates ((El Hariri El Nokab and van der Wel 2020)), RNA (Ahmed et al 2020) or membranes (Dufourc 2021),(Mallikarjunaiah et al 2019) to larger systems such as protein complexes(Demers et al 2018) large proteins (Vasa et al 2018)(Schütz 2021)), protein-ligand interaction ((Vasa et al 2019)(Elkins and Hong 2019),(Medeiros-Silva et al 2019)), misfolded proteins (König et al 2019), amyloid (Tycko 2016)fibrils ((Jaroniec 2019),(Loquet et al 2018)), helical filaments (Habenstein et al 2019), viruses (Lecoq et al 2020), (Gupta et al 2020), (Lu et al 2020)), membrane proteins (McDermott 2009)(Tang et al 2013)(Mandala et al 2018) or whole cells ((Narasimhan et al 2020)). In the two past decades, structural investigation of biomolecules at atomic resolution by SSNMR has made dramatic analytical improvements with the introduction of direct proton ( 1 H) detection(Ishii et al 2001)(Reif et al 2001)(Paulson et al 2003)(Zhou et al 2009) combined with the use of magic-angle spinning (MAS) probes operating at fast frequencies(Nishiyama 2016)(Böckmann et al 2015)(Cala-De Paepe et al 2017)(Sternberg et al 2018)(Xue et al 2018)(Ishii et al 2018)(Schledorn et al 2020), nowadays commercially available at MAS frequency of 60-110 kHz.…”