Protein sample preparation for solid-state NMR investigations

doi:10.1016/j.pnmrs.2019.01.001

Progress in Nuclear Magnetic Resonance Spectroscopy

Volume 110, February 2019, Pages 20-33

https://doi.org/10.1016/j.pnmrs.2019.01.001 Get rights and content

Highlights

•
Sample preparation is central in solid-state NMR in order to analyze proteins.
•
New solid-state NMR methods allow to address large and complex (membrane) proteins samples.
•
Selective labeling techniques become central to resolve the resonances.
•
Smaller and smaller amounts of protein are needed with faster magic-angle spinning.
•
In this context, cell-free expression becomes a promising tool for sample preparation.

Abstract

Preparation of a protein sample for solid-state NMR is in many aspects similar to solution-state NMR approaches, mainly with respect to the need for stable isotope labeling. But the possibility of using solid-state NMR to investigate membrane proteins in (native) lipids adds the important requirement of adapted membrane-reconstitution schemes. Also, dynamic nuclear polarization and paramagnetic NMR in solids need specific schemes using metal ions and radicals. Sample sedimentation has enabled structural investigations of objects inaccessible to other structural techniques, but rotor filling using sedimentation has become increasingly complex with smaller and smaller rotors, as needed for higher and higher magic-angle spinning (MAS) frequencies. Furthermore, solid-state NMR can investigate very large proteins and their complexes without the concomitant increase in line widths, motivating the use of selective labeling and unlabeling strategies, as well as segmental labeling, to decongest spectra. The possibility of investigating sub-milligram amounts of protein today using advanced fast MAS techniques enables alternative protein synthesis schemes such as cell-free expression. Here we review these specific aspects of solid-state NMR sample preparation.

Graphical abstract

Introduction

Solid-state NMR is one of the more recent arrivals in the field of biophysical structure analysis techniques. Solid-state and solution-state NMR were actually developed about at the same time, but the application to solid biomolecules was for long hampered by the presence of broad resonance lines, which restricted the information attainable for large multi-spin systems like proteins. Solution-state NMR was thus the first approach developed in this context [1], and protein solid-state NMR emerged only when NMR technology had advanced to provide static fields high enough and magic-angle spinning fast enough to obtain sufficient resolution and sensitivity to detect and resolve the many spins in a protein. Another concomitant factor was that the cost of recombinant production and isotope labeling of proteins was coming down to affordable ranges, enabling production of the several tens of milligrams ¹³C/¹⁵N labeled samples the method required.

Solid-state NMR sample preparation is of central importance in protein studies, as also for solution-state NMR, x-ray crystallography, and cryo-electron microscopy (EM). In contrast to EM, which also can analyze samples extracted from organisms, for NMR the proteins to be studied most often need to be produced recombinantly, since isotope labeling is of high importance. In solid-state NMR, labeling may be even more important, as solid-state homonuclear ¹H spectroscopy remains difficult with current proton linewidths, even under fast MAS. Also, probe and rotor background signals can add difficulties to the detection of the desired protein signals.

Solid-state NMR has the strength to go beyond the characterization of relatively small and soluble biomolecules, and can analyze large assemblies and complexes thereof. Sample preparation thus often goes beyond the expression and purification of the protein, and includes further steps like membrane reconstitution, assembly, or addition of interacting partners. The approaches thus share aspects with sample preparation for electron microscopy [2], [3], [4]. However, unlike for EM, the sample is often hydrated and at room temperature during the NMR experiment, conditions which might be considered closer to physiological.

Solid-state NMR needs homogenous protein samples, as measured linewidths are directly related to the local order the proteins show in the samples. Highest quality NMR spectra are still obtained on highly ordered assemblies, like small, rigid crystalline proteins, or highly rigid assemblies like bacterial needles or virus capsids. Understanding of how exactly the linewidth is related to order and (residual) dynamics is slowly emerging, but more work is needed to gain deeper insight.

Solid-state NMR was previously infamous for the high sample amounts it needed. The field took off twenty years ago with the use of sample containers (rotors) which needed about 40–50 mg starting material to be optimally filled. The latest developments today reduce this amount to below 0.5 mg, i.e. by a factor of 100. This opens perspectives which were unthought-of before, mainly in terms of types of proteins that can be analyzed. In terms of sample requirement, this actually puts solid-state NMR in one of the best positions in the panel of available high-resolution biophysical structure analysis methods. If EM can in principle analyze samples extracted from organisms, purity is there also of concern, and sorting of polymorphs, for example, is not always straightforward with current resolution, even if progress is highly promising. It is particularly interesting to combine solid-state NMR with cell-free expression, as there only the protein to be analyzed is “visible” as it represents the only isotope labeled material, and any additives/interacting partners will remain invisible in the spectra. The important drop in the required sample amount opens the possibility for this combination today.

We will review here sample preparation methods which have been used for solid-state NMR, but which often are derived from other biophysical methods, or which can be used by other methods as well. We will start from protein production by cellular methods for the introduction of selective labels, and cell-free methods; describe membrane protein reconstitution into lipids, as well as sample preparation for DNP and paramagnetic NMR. We finally review the basics of protein sedimentation which is today the main principle of sample preparation for solid-state NMR.

Section snippets

Residue-selective isotope labeling

Bacterial expression remains the workhorse for NMR sample preparation, and has been described extensively in the literature [5]. Selective labeling has been used in solution NMR, with a focus recently on methyl-group labeling in the context of the study of large proteins and the application of TROSY-based NMR approaches [6]. In contrast to solution NMR, where linewidths are dependent on rotational correlation times and thus protein size, which hampers analysis of large proteins or complexes,

Segmental labeling

Segmental isotope labeling of individual domains in a full-length construct allows the study of domains of a large protein separately. This is particularly useful to simplify the resonance assignments of large proteins in the solid state or to study the interaction of an individual domain within a protein-protein complex, with RNA and DNA or other small molecules. In such an approach, one of the two domains is made visible for NMR by ¹³C and ¹⁵N isotope labeling. This domain is ligated with a

Cell-free protein synthesis

Bacterial expression as described above, and more generally cell-based expression, is not always the method of choice for sample preparation for NMR studies, especially in the case of eukaryotic membrane proteins. Indeed, although some have been successfully expressed in cell-based expression systems, e.g. in Lactococcus lactis [23], in human [24], [25] or in insect cells [26], the production of such proteins is still challenging. Overexpression is often impaired by toxicity of the target

Membrane protein reconstitution

One of the major advantages of solid-state NMR is the possibility to conduct structural investigations on membrane proteins inserted in a lipid environment. This feature is important because many membrane proteins display their full activity only when embedded in a lipid bilayer. Therefore, the reconstitution in lipids procedure can be considered as a cornerstone in the sample preparation. The reconstitution process is based on the co-micellisation of purified membrane protein in detergent with

Dynamic nuclear polarization (DNP)

DNP is a sensitivity-enhancement method based on transferring the much larger electron polarization to surrounding nuclei [111], [112], in particular via electron-proton two- or three-spin processes and followed by ¹H–¹H spin-diffusion [113]. This is achieved by irradiating the sample with a microwave source and saturating the EPR lines of added organic radicals. This approach has been used in NMR, in biology and materials [114], [115], [116], [117], [118]. Protein DNP requires specific sample

Paramagnetic NMR

Paramagnetic NMR is a well-established tool in solution-state NMR, where for example it is used to identify interaction sites like nucleotide binding sites and to derive structural restraints. For reviews see Refs. [135], [136]. Paramagnetic relaxation enhancements (PREs) and Pseudo Contact Shifts (PCS) are used in protein structure determination [135]. The use of paramagnetic effects in biomolecular solid-state NMR developed over the last years [137], [138], [139], [140], and has been used to

Sedimentation and rotor filling

An important step in sample preparation is the filling of the protein into the NMR rotor. As the protein is subject to high centrifugal forces in the rotor, the best approach is to use ultracentrifugation to fill the rotor, as this avoids void space once the rotor is spun in the NMR probe. For this, special filling tools have been developed over recent years by different groups [109], [151], [152] (see also Fig. 9), and commercial filling tools are now also available. Concomitant with rotor

Summary and outlook

Sample preparation is progressing from being the compulsory first (sometimes difficult and, for many NMR spectroscopists, still mysterious and annoying) step to becoming a real tool box, capable of adapting proteins to spectroscopic needs, potentials and limitations. Singling out certain protein domains, amino acid types or pairs thereof by specific labeling, playing with deuteration and protonation patterns, simply sedimenting proteins of interest, and most importantly synthesizing proteins

Acknowledgements

This work was supported by the French Agence Nationale de Recherche (ANR-14-CE09-0024B), the LABEX ECOFECT (ANR-11-LABX-0048) within the Université de Lyon program Investissements d’Avenir (ANR-11-IDEX-0007), and by the Swiss National Science Foundation (Grant 200020_159707). T.W. acknowledges support by the ETH Career SEED-69 16-1.

Glossary

ABC: ATP-binding cassette
AMP-PNP: Adenylyl-imidodiphosphate
cDNA: complementary DNA
CECF: continuous-exchange CF system
CF: cell-free
CFCF: continuous flow CF
CMC: critical micelle concentration
CTD: C-terminal domain
Cryo-EM: cryo-electron microscopy
DARR: dipolar-assisted rotational recoupling
DNA: deoxyribonucleic acid
DNP: dynamic nuclear polarization
E. coli: Escherichia coli
FUS: fused in sarcoma
GRecon: Gradient reconstitution
HpDnaB: Helicobacter pylori DnaB
LPR: lipid-to-protein ratio
NMR: nuclear magnetic resonance
NTD: N-terminal domain

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Protein sample preparation for solid-state NMR investigations

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Residue-selective isotope labeling

Segmental labeling

Cell-free protein synthesis

Membrane protein reconstitution

Dynamic nuclear polarization (DNP)

Paramagnetic NMR

Sedimentation and rotor filling

Summary and outlook

Acknowledgements

Glossary

Curr. Opin. Struct. Biol.

Structure

Biochim. Biophys. Acta

Protein Expr Purif.

Protein Expr Purif.

Protein Expr Purif.

Meth. Enzymol.

Biochim. Biophys. Acta (BBA) - Biomemb.

Protein Expr Purif.

Meth. Enzymol.

J. Biol. Chem.

Trends Biotechnol.

Protein Expr. Purif.

N. Biotechnol.

Curr. Opin. Biotechnol.

FEBS Lett.

FEBS Lett.

Trends Biotechnol.

J. Biol. Chem.

J. Biol. Chem.

Protein Expr Purif.

J. Biol. Chem.

J. Biol. Chem.

Progr. NMR Spectr.

Protein Expr Purif.

Curr. Opin. Struct. Biol.

Meth. Enzymol.

Curr. Opin. Struct. Biol.

Biochim. Biophys. Acta

NMR Studies of structure and function of biological macromolecules (Nobel Lecture)

Angew. Chem. Int. Ed.

Cryo-EM structure of alpha-synuclein fibrils

Elife

GRecon: a method for the lipid reconstitution of membrane proteins

Angew. Chem. Int. Ed.

Gradient reconstitution of membrane proteins for solid-state NMR studies

J. Biomol. NMR

Recombinant protein expression in E. coli: a historical perspective

Methods Mol. Biol.

Bacterial metabolism

Annu. Rev. Microbiol.

Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy

J. Biomol. NMR

Synthesis of proteins by native chemical ligation

Science

Expressed protein ligation: a general method for protein engineering

Proc. Natl. Acad. Sci. USA

Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803

Proc. Natl. Acad. Sci. USA

Protein trans-splicing and its use in structural biology: opportunities and limitations

Mol. BioSyst.

Sortase-mediated protein ligation: a new method for protein engineering

JACS

Untangling a repetitive amyloid sequence: correlating biofilm-derived and segmentally labeled Curli Fimbriae by solid-state NMR spectroscopy

Angew. Chem. Int. Ed.

Unambiguous assignment of short- and long-range structural restraints by solid-state NMR spectroscopy with segmental isotope labeling

Chembiochem.

Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR

J. Biomol. NMR

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Proc. Natl. Acad. Sci. USA

Segmental isotope labelling and solid-state NMR of a 12 × 59 kDa motor protein: identification of structural variability

J. Biomol. NMR

Structure and primase-mediated activation of a bacterial dodecameric replicative helicase