Assigning methyl resonances for protein solution-state NMR studies

Highlights

• Methyl labeling allows NMR studies of large proteins.

• Methyl groups can be assigned in the presence and absence of backbone NMR data.

• Isotope labeling strategies for all methyl containing amino acids exist.

• 3D and 4D NOESY methods can be optimized for methyl assignments.

• Automated data analysis facilitates methyl assignments.

Abstract

Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has substantially expanded the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to grow, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (>50 kDa) proteins. In this practical review, we present strategies to efficiently isotopically label proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis. We use our experiences from assigning methyl resonances for the aromatic biosynthetic enzymes tryptophan synthase and chorismate mutase to provide advice for all stages of experimental set-up and data analysis.

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.