REDOR recoupling in polymer-stabilized liquid crystals undergoing MAS — Two-dimensional NMR applications with strongly aligned proteins

Abstract

We recently introduced a technique, termed SAD-REDOR, for recoupling residual dipolar couplings in a single-domain polymer-stabilized liquid crystal (PSLC) undergoing magic-angle spinning (MAS). The experiment was demonstrated with 1H–15N dipolar couplings in a small molecule. Here, we show the applicability of the technique to a strongly aligned protein and we describe a novel two-dimensional experiment to generate in-phase and anti-phase (IPAP) doublets in the 1H dimension of an HSQC NMR spectrum. This pulse sequence, SAD-IPAP, was tested on a sample of 15N-labeled ubiquitin (5 mmol/L) in a polyacrylamide-stabilized Pf1 phage liquid crystal (20 mg/mL). 15N–1H residual dipolar couplings (RDCs) were measured with the SAD-IPAP pulse sequence at spinning speeds of 1000 and 1250 Hz. RDSs were also measured using the conventional HSQC-IPAP method in a sample of 15N-ubiquitin dissolved in a solution of Pf1 phage (1 mg/mL). The resulting RDCs were fitted to the solution structure of ubiquitin to estimate the alignment tensor and to determine the accuracy of the measured couplings. The results highlight the benefits of SAD-REDOR for the measurement of RDCs in strongly aligned macromolecules.Key words: residual dipolar couplings (RDCs), polymer-stabilized liquid crystals (PSLCs), rotational echo double resonance (REDOR) recoupling, magic-angle spinning (MAS), ubiquitin, biomolecular nuclear magnetic resonance (NMR), proteins.