A methyl-TROSY approach for NMR studies of high-molecular-weight DNA with application to the nucleosome core particle

Abstract

The development of methyl-transverse relaxation-optimized spectroscopy (methyl-TROSY)–based NMR methods, in concert with robust strategies for incorporation of methyl-group probes of structure and dynamics into the protein of interest, has facilitated quantitative studies of high-molecular-weight protein complexes. Here we develop a one-pot in vitro reaction for producing NMR quantities of methyl-labeled DNA at the C5 and N6 positions of cytosine (5mC) and adenine (6mA) nucleobases, respectively, enabling the study of high-molecular-weight DNA molecules using TROSY approaches originally developed for protein applications. Our biosynthetic strategy exploits the large number of naturally available methyltransferases to specifically methylate DNA at a desired number of sites that serve as probes of structure and dynamics. We illustrate the methodology with studies of the 153-base pair Widom DNA molecule that is simultaneously methyl-labeled at five sites, showing that high-quality13C-1H spectra can be recorded on 100 μM samples in a few minutes. NMR spin relaxation studies of labeled methyl groups in both DNA and the H2B histone protein component of the 200-kDa nucleosome core particle (NCP) establish that methyl groups at 5mC and 6mA positions are, in general, more rigid than Ile, Leu, and Val methyl probes in protein side chains. Studies focusing on histone H2B of NCPs wrapped with either wild-type DNA or DNA methylated at all 26 CpG sites highlight the utility of NMR in investigating the structural dynamics of the NCP and how its histone core is affected through DNA methylation, an important regulator of transcription.

A quantitative model predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions

“…The population was ~2-fold higher than the value measured in the nucleobase in organic solvent ( Fig. 1a) 25 while the exchange rate was ~20-fold faster, and in better agreement with values reported recently for ssDNA 34 (at T = 45°C, Supplementary Table 1). Similar syn-anti isomerization kinetics were obtained for another different sequence (Extended Data Fig.…”

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

“…For proteins under 20 kDa, RD experiments can be recorded on protonated 15 N-labeled samples, whereas deuteration and transverse relaxation optimized spectroscopy (TROSY; Pervushin et al 1997 ) are required for larger systems. In very large complexes (> 80 kDa), methyl group labeling in a fully deuterated background combined with methyl TROSY based approaches (Tugarinov et al 2003 ; Schütz and Sprangers 2019 ; Abramov et al 2020 ) can be used to record 13 C single quantum (SQ; Skrynnikov et al 2001 ; Lundström et al 2007 ; Rennella et al 2016 ) and 13 C multiple quantum (MQ; Korzhnev et al 2004 ) as well as 1 H SQ (Tugarinov and Kay 2007 ; Baldwin et al 2010 ; Otten et al 2010 ; Weininger et al 2012 ), double quantum (DQ; Gopalan et al 2018 ) and triple quantum (TQ; Gopalan et al 2018 ) RD profiles.…”

Analysis of Conformational Exchange Processes using Methyl-TROSY-Based Hahn Echo Measurements of Quadruple-Quantum Relaxation

“…The study of methyl groups is of fundamental importance to modern biomolecular NMR spectroscopy, both in proteins and in labelled nucleic acids [52][53][54] . Methyl 13 CH3 spin systems provide a complex set of energy levels (Fig.…”