Assigning methyl resonances for protein solution-state NMR studies
Introduction
NMR has provided critical insights into protein structure, dynamics and function. Solution-state NMR has a protein size limitation owing to the relationship between the slow tumbling of large proteins in solution and its effects on relaxation properties and signal acquisition. Several advancements over many years have lifted protein size limitations, including the development of methods to analyze 1H–13C methyl resonances [1], [2], [3], [4]. These studies have allowed solution-state NMR studies to be carried out on proteins and complexes several hundred kilodaltons to up to 1 megadalton in size [5], [6], [7], [8], [9].
Methyl labeling offers several advantages over 1H–15N backbone amide labeling schemes for large proteins. Methyl protons have threefold degeneracy due to rapid rotation on the methyl axis, meaning that all three protons contribute to the same NMR signal [3]. Methyl groups also have enhanced sensitivity for any HMQC (heteronuclear multiple quantum coherence) type experiments. HMQC experiments have a field-independent transverse relaxation optimized (TROSY) effect for methyl groups, and so provide signals with narrow linewidths even for proteins >100 kDa [3]. Additionally, the 1H–13C methyl NMR spectrum will often have fewer overlapping peaks compared to 15N backbone labeling because on average only 30% of the amino acids in proteins contain methyl groups [10].
Methyl-containing amino acids are well-distributed throughout the protein structure, and so they are excellent probes for structural characterization of large proteins. Consistent with the theme of the present issue, there are also several NMR methods to gain insight into biomolecular structural dynamics across many timescales. Experiments for studying events on the microsecond-to-second timescale such as DEST (Dark state Exchange Saturation Transfer) [11], CEST (Chemical Exchange Saturation Transfer) [12], [13], [14], CPMG (Carr-Purcell-Meiboom-Gill) relaxation-dispersion [15], [16], [17], and paramagnetic relaxation enhancement (PRE) [18] have been developed for methyl groups. The sensitivity of methyl groups is also useful for studying dynamics by line shape analysis involving micromolar affinity ligand binding since expedient 2D correlation spectra can be obtained even on low (<50 µM) concentration protein samples in as short a time as 10 min [19].
There are several reviews that explore the merits of protein methyl NMR and the scientific insights resulting from these studies (e.g. [7], [9], [20]). Here, we focus on perhaps the rate-limiting step of these studies – the initial assignment of the methyl resonances. We use our own experiences with enzymes from the aromatic biosynthetic pathway, namely tryptophan synthase and chorismate mutase, to guide the reader through this process. For tryptophan synthase, we studied the ∼30 kDa alpha subunit (αTS), where we could base the 1H–13C methyl assignments on the previously established 1H–15N amide backbone resonance assignments [21], [22]. For the ~60 kDa chorismate mutase, there were no previous NMR assignments, and so we used a NOESY (Nuclear Overhauser Effect SpectroscopY) based method [23] along with a known X-ray crystal structure [24] to determine assignments. We have also previously assigned methyl resonances for the 52 kDa poliovirus RNA-dependent RNA polymerase [25] through mutagenesis (i.e. look for the disappearance of a resonance after changing a methyl bearing residue to a different residue that will not be similarly isotopically labeled), but this method can be tedious, potentially very expensive and assignments are not always conclusive, although there have been efforts to streamline this approach [26]. We note that there are other exciting, perhaps complementary methods, that take advantage of paramagnetic probes to assign methyl groups [27], [28], but we do not go into detail with those strategies here. In this Review, we will discuss different isotopic labeling schemes and their merits, and practical considerations for NMR data acquisition, processing and analysis for methyl assignments. We first discuss assigning methyl resonances based on previously acquired 1H–15N amide backbone assignments, and then have a deeper discussion on methyl assignment strategies in the absence of backbone assignment data based on NOESY methods.
Section snippets
Assigning side chain methyl resonances based on previous backbone assignments
For smaller proteins (<20 kDa), methyl resonances can often be assigned based on backbone resonance assignments (following classical 1H/13C/15N triple resonance experiments [29], [30], [31], [32]) using TOCSY (total correlation spectroscopy) type transfer along the aliphatic side chain [33], [34]. However, the slow overall tumbling times for larger proteins lead to diminished magnetization during the TOCSY mixing period. The presence of 1H groups near methyl side chain groups of interest can
Assigning side chain methyl resonances in the absence of backbone assignments
Methyl resonances can also be assigned in the absence of any backbone resonance assignments. This strategy may be especially important for larger proteins (>50 kDa), where it can be very challenging to assign backbone resonances using 1H, 13C, 15N triple resonance methods. One strategy is to assign methyl resonances using NOESY methods, taking advantage of through-space nuclear spin polarization transfer via cross-relaxation between NMR active nuclei. The NOE pattern predicted by a 3D protein
Summary
Solution-state NMR experiments on methyl groups have brought great insight into the structure and function of proteins. Here, we have used our experiences with assigning the methyl resonances of aromatic amino acid biosynthetic enzymes to help guide similar studies on other proteins of interest. Further advances in pulse sequence development, data analysis and alternative methods may make methyl assignments possible for even larger protein complexes or proteins under less optimal conditions
Acknowledgments
The authors would like to thank Dr. Jinfa Ying for providing the methyl NOESY pulse sequence and for help setting up the methyl NOESY experiment and SMILE scripts, Dr. Frank Delaglio for technical support with NMRpipe and Dr. Andrew Baldwin for help with MAGMA. The authors would also like to thank Dr. Erik Cook, Rebecca D’Amico, Grace Usher and Dennis Winston for feedback on this manuscript. This work was supported by the National Science Foundation [MCB-1615032]; and the National Institutes of
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Allostery, engineering and inhibition of tryptophan synthase
2023, Current Opinion in Structural BiologyUtility of methyl side chain probes for solution NMR studies of large proteins
2023, Journal of Magnetic Resonance OpenConformational Transitions in Yeast Chorismate Mutase Important for Allosteric Regulation as Identified by Nuclear Magnetic Resonance Spectroscopy
2022, Journal of Molecular BiologyCitation Excerpt :To complement our previous analysis43 and to gain additional insight into the conformational states present in solution, we evaluated NMR chemical shift differences for Ile and Thr methyl groups between ScCM in the absence of allosteric effector (ScCM:Apo) and in the presence of saturating Tyr (ScCM:YY) or saturating Trp (ScCM:WW). The 1H–13C resonances for the δ1 Ile and γ2 Thr methyl groups have been previously assigned.50 It is expected that ScCM:YY and ScCM:WW should be primarily present as the T and R conformations respectively, based on X-ray crystal structures36–37 and binding constants previously determined from ITC.18,43
Emerging solution NMR methods to illuminate the structural and dynamic properties of proteins
2019, Current Opinion in Structural BiologyCitation Excerpt :In this section we will discuss the recent developments in methods for resonance assignment and analysis of structure and dynamics. For the assignment of methyl groups, besides brute force mutation-based strategies, methods to correlate methyl resonances to their corresponding main-chain 15N and 13Cα chemical shifts have been previously proposed [30–32,33•]. More recently, Kerfah et al. published a convenient method to establish CH3-specific NMR assignment of Ala, Ile, Leu, and Val methyl groups in high molecular weight proteins using a single sample [34].
Toolkit for NMR Studies of Methyl-Labeled Proteins
2019, Methods in EnzymologyCitation Excerpt :These combined technical advances have allowed unprecedented atomic level understanding in system that had remained impervious to structural characterization by crystallography (Saio, Guan, Rossi, Economou, & Kalodimos, 2014). In spite of the advances in the field, aspects of methyl assignment and reagent cost still pose a challenge to routine implementation in structural biology laboratories (Alphonse & Ghose, 2018; Gorman, Debashish, O'Rourke, & Boehr, 2018). A schematic approach to methyl NMR spectroscopy of biomolecules is outlined in Fig. 1.