Specific isotopic labelling and reverse labelling for protein NMR spectroscopy: using metabolic precursors in sample preparation.

The study of protein structure, dynamics and function by NMR spectroscopy commonly requires samples that have been enriched ('labelled') with the stable isotopes 13C and/or 15N. The standard approach is to uniformly label a protein with one or both of these nuclei such that all C and/or N sites are in principle 'NMR-visible'. NMR spectra of uniformly labelled proteins can be highly complicated and suffer from signal overlap. Moreover, as molecular size increases the linewidths of NMR signals broaden, which decreases sensitivity and causes further spectral congestion. Both effects can limit the type and quality of information available from NMR data. Problems associated with signal overlap and signal broadening can often be alleviated though the use of alternative, non-uniform isotopic labelling patterns. Specific isotopic labelling 'turns on' signals at selected sites while the rest of the protein is NMR-invisible. Conversely, specific isotopic unlabelling (also called 'reverse' labelling) 'turns off' selected signals while the rest of the protein remains NMR-visible. Both approaches can simplify NMR spectra, improve sensitivity, facilitate resonance assignment and permit a range of different NMR strategies when combined with other labelling tools and NMR experiments. Here, we review methods for producing proteins with enrichment of stable NMR-visible isotopes, with particular focus on residue-specific labelling and reverse labelling using Escherichia coli expression systems. We also explore how these approaches can aid NMR studies of proteins.

Optimized precursor to simplify assignment transfer between backbone resonances and stereospecifically labelled valine and leucine methyl groups: application to human Hsp90 N-terminal domain.

Methyl moieties are highly valuable probes for quantitative NMR studies of large proteins. Hence, their assignment is of the utmost interest to obtain information on both interactions and dynamics of proteins in solution. Here, we present the synthesis of a new precursor that allows connection of leucine and valine pro-S methyl moieties to backbone atoms by linear 13C-chains. This optimized 2H/13C-labelled acetolactate precursor can be combined with existing 13C/2H-alanine and isoleucine precursors in order to directly transfer backbone assignment to the corresponding methyl groups. Using this simple approach leucine and valine pro-S methyl groups can be assigned using a single sample without requiring correction of 1H/2H isotopic shifts on 13C resonances. The approach was demonstrated on the N-terminal domain of human HSP90, for which complete assignment of Ala-ß, Ile-δ1, Leu-δ2, Met-ε, Thr-γ and Val-γ2 methyl groups was obtained.

In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies.

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membrane proteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex.

Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.

Unraveling self-assembly pathways of the 468-kDa proteolytic machine TET2.

The spontaneous formation of biological higher-order structures from smaller building blocks, called self-assembly, is a fundamental attribute of life. Although the protein self-assembly is a time-dependent process that occurs at the molecular level, its current understanding originates either from static structures of trapped intermediates or from modeling. Nuclear magnetic resonance (NMR) spectroscopy has the unique ability to monitor structural changes in real time; however, its size limitation and time-resolution constraints remain a challenge when studying the self-assembly of large biological particles. We report the application of methyl-specific isotopic labeling combined with relaxation-optimized NMR spectroscopy to overcome both size- and time-scale limitations. We report for the first time the self-assembly process of a half-megadalton protein complex that was monitored at the structural level, including the characterization of intermediate states, using a mutagenesis-free strategy. NMR was used to obtain individual kinetics data on the different transient intermediates and the formation of final native particle. In addition, complementary time-resolved electron microscopy and native mass spectrometry were used to characterize the low-resolution structures of oligomerization intermediates.

Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3 labelling: application to the 50S ribosome subunit.

Solid-state NMR spectroscopy allows the characterization of the structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths increase only marginally. We apply this labelling strategy to a 468 kDa-large dodecameric aminopeptidase, TET2, and the 1.6 MDa-large 50S ribosome subunit of Thermus thermophilus.

CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample.

A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back (13)C-(13)C TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu) substantially compromises the efficiency of the TOCSY transfers. To permit the use of this efficient pulse scheme, a series of optimized precursors were designed to yield linear (13)C perdeuterated side-chains with a single protonated CH3 group in these three residues. These precursors were added to the culture medium for incorporation into expressed proteins. For Val and Leu residues, the topologically different spin-systems introduced for the pro-R and pro-S methyl groups enable stereospecific assignment. All CH3 can be simultaneously assigned on a single sample using a TOCSY experiment. It only requires the tuning of a mixing delay and is thus more versatile than the relayed COSY experiment. Enhanced resolution and sensi-tivity can be achieved by non-uniform sampling combined with the removal of the large JCC coupling by deconvolution prior to the processing by iterative soft thresholding. This strategy has been used on malate synthase G where a large percentage of the CH3 groups could be correlated directly up to the backbone Ca. It is anticipated that this robust combined strategy can be routinely applied to large proteins.

Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins.

Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the development of new isotopic labeling strategies has opened the possibility of exploiting NMR spectroscopy in the study of supra-molecular complexes with molecular weights of up to 1MDa. At the core of these isotopic labeling developments is the specific introduction of [(1)H,(13)C]-labeled methyl probes into perdeuterated proteins. Here, we describe the evolution of these approaches and discuss their impact on structural and biological studies. The relevant protocols are succinctly reviewed for single and combinatorial isotopic-labeling of methyl-containing residues, and examples of applications on challenging biological systems, including high molecular weight and membrane proteins, are presented.

Scrambling free combinatorial labeling of alanine-ß, isoleucine-δ1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs.

Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L and V are stereo-specifically labeled. We show that 2-ketobutyrate cannot be combined with Ala and 2-acetolactate (for the stereo-specific labeling of L and V) as this results in co-incorporation incompatibility and isotopic scrambling. Thus, we developed a robust and cost-effective enzymatic synthesis of the isoleucine precursor, 2-hydroxy-2-(1'-[(2)H2], 2'-[(13)C])ethyl-3-keto-4-[(2)H3]butanoic acid, as well as an incorporation protocol that eliminates metabolic leakage. We show that application of this labeling scheme to a large 82 kDa protein permits the detection of long-range (1)H-(1)H NOE cross-peaks between methyl probes separated by up to 10 Å.

A cost-effective protocol for the parallel production of libraries of 13CH3-specifically labeled mutants for NMR studies of high molecular weight proteins.

There is increasing interest in applying NMR spectroscopy to the study of large protein assemblies. Development of methyl-specific labeling protocols combined with improved NMR spectroscopy enable nowadays studies of proteins complexes up to 1 MDa. For such large complexes, the major interest lies in obtaining structural, dynamic and interaction information in solution, which requires sequence-specific resonance assignment of NMR signals. While such analysis is quite standard for small proteins, it remains one of the major bottlenecks when the size of the protein increases. Here, we describe implementation and latest improvements of SeSAM, a fast and user-friendly approach for assignment of methyl resonances in large proteins using mutagenesis. We have improved culture medium to boost the production of methyl-specifically labeled proteins, allowing us to perform small-scale parallel production and purification of a library of (13)CH3-specifically labeled mutants. This optimized protocol is illustrated by assignment of Alanine, Isoleucine, and Valine methyl groups of the homododecameric aminopeptidase PhTET2. We estimated that this improved method allows assignment of ca. 100 methyl cross-peaks in 2 weeks, including 4 days of NMR time and less than 2 k€ of isotopic materials.