Measurement of residual dipolar couplings in methyl groups via carbon detection

Abstract

Residual dipolar couplings (RDCs) provide both structural and dynamical information useful in the characterization of biological macromolecules. While most data come from the interaction of simple pairs of directly bonded spin-1/2 nuclei ( 1 H-15 N, 1 H-13 C, 1 H-1 H), it is possible to acquire data from interactions among the multiple spins of 13 C-labeled methyl groups ( 1 H 3 -13 C). This is especially important because of the advantages that observation of 13 C-labled methyl groups offers in working with very large molecules. Here we consider some of the options for measurement of methyl RDCs in large and often fully protonated proteins and arrive at a pulse sequence that exploits both J-modulation and direct detection of 13 C. Its utility is illustrated by application to a fully protonated two domain fragment from the mammalian glycoprotein, Robo1, 13 C-methyllabeled in all valines.

Murine norovirus capsid plasticity – Glycochenodeoxycholic acid stabilizes P-domain dimers and triggers escape from antibody recognition

“…The A’B’ loop should also undergo conformational reorganization upon GCDCA binding but is lacking 13 C-methyl reporter groups. Future work, e.g., based on measurements of residual 13 C- 1 H dipolar couplings (52, 53) may complete the overall picture. The plasticity and conformational changes in the surface loops triggered by bile acid represent a novel allosteric immune escape pathway for murine noroviruses.…”

13C Direct Detected NMR for Challenging Systems

“…The resolved 13 C′– 13 C α scalar couplings are thus an obvious candidate, easily accessible from carbonyl detected experiments, to determine RDCs. Experiments to measure a variety of different RDCs, including several that involve proton nuclei, ,, were developed to enable the determination of RDCs also in cases in which methods based on 1 H direct detection fails to provide information. , …”

Opportunities and Challenges of Backbone, Sidechain, and RDC Experiments to Study Membrane Protein Dynamics in a Detergent-Free Lipid Environment Using Solution State NMR

“…New pulse sequences, such as methyl 13 C- or 1 H-CEST even allow to quantify populations, interconversion kinetics and structural features of sparsely populated “invisible” protein states and holds great promise to investigate lowly populated “excited” membrane protein conformations (Vallurupalli et al, 2012, 2017). The applicability of methyl RDCs (Sprangers and Kay, 2007a; Williams et al, 2019) for the investigation of membrane protein dynamics in 100+ kDa assemblies remains to be seen. Nevertheless, all these new developments provide exciting new opportunities to investigate membrane protein dynamics in a more natural lipid environment on a wide range of timescales.…”

“…Second, the accuracy of RDCs measured from rather large assemblies (close to or above 100 kDa) remains another major challenge. It is therefore conceivable that methyl RDCs could play a more dominant role in the future in assessing dynamics of membrane proteins in a detergent-free environment (Sprangers and Kay, 2007a; Williams et al, 2019). Their sharper signals due to their flexibility and their higher signal intensity due to 3 hydrogens per group will lead to superior RDC accuracy and quality when compared to backbone 1 H- 15 N RDCs.…”