This work is funded by the ANR (13-PDOC-0017-01 to B.H. and ANR-14-CE09-0020-01 to A.L.), &#34;Investments for the future&#34; Programme IdEx Bordeaux/CNRS (PEPS 2016 to B.H.) reference ANR-10-IDEX-03-02 to B.H., the Fondation pour la Recherche M&#233;dicale (FRM-AJE20140630090 to A.L.), the FP7 program (FP7-PEOPLE-2013-CIG to A.L.) and the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (ERC Starting Grant to A.L., agreement No 639020) and project &#34;WEAKINTERACT.&#34;

The authors have nothing to disclose.

Solid-state NMR (SSNMR) is a method of choice for characterizing macromolecular protein assemblies at an atomic level. One of the central issues in SSNMR-based structure determination is the spectral quality of the investigated system, that allows establishing 3D structural models of various precision, typically ranging from low-resolution models (containing the secondary structure elements and little 3D information) to pseudo-atomic 3D structures. The quantity and quality of structural information extracted from multi-dimensional SSNMR experiments is the key to compute a high-resolution NMR structure of the assembly.
The described protocol relies on the detection of 13C-13C and 15N-13C structural restraints requiring the recording of several 2D (and sometimes 3D) spectra with high signal-to-noise. At moderate MAS frequencies (&#60;25 kHz), the sample is introduced into rotors with sizes of 3.2-4 mm diameter allowing for protein quantities of up to ~50 mg, dependent on the sample hydration. The amount of sample inside the rotor is directly proportional to the signal-to-noise ratio in SSNMR spectra, a decisive factor for the detection of long-range distance restraints and their unambiguous assignment.
The spectral resolution is a crucial parameter during the sequential resonance assignment and the restraints collection. To obtain optimal results, the sample preparation parameters need to be optimized, particularly in the purification of the subunit and the assembly conditions (pH, buffer, shaking, temperature, etc.). For sample optimization, it is recommended to prepare unlabeled samples for several distinct conditions for which assembly has been observed, and to record a 1D 1H-13C CP spectrum (described in step 2.1) on each prepared sample. The spectra serve to compare spectral resolution and dispersion between the different preparations, based upon which the optimal conditions can be determined.
The quality of the SSNMR data depends strongly on the choice of the NMR acquisition parameters, especially for the polarization transfer steps. The use of high magnetic field strengths (&#8805;600 MHz 1H frequency) is essential for high sensitivity and spectral resolution, required when facing complex targets such as macromolecular protein assemblies.
A limiting factor in many cases is the spectrometer availability. Therefore, a judicious choice of the samples to be prepared should precede the spectrometer session. In any case, a uniformly 13C, 15N-labeled sample is a prerequisite to perform the sequential and intra-residual resonance assignment. For proteins assigned by solid-state NMR techniques see71. Structure determination of macromolecular assemblies at moderate MAS frequencies requires selectively 13C-labeled samples; for the detection of long-range 13C-13C and 13C-15N contacts samples based on 1,3-13C- and 2-13C-gylcerol and/or 1-13C - and 2-13C-glucose labeling are commonly used, as described above. The choice between the two labeling schemes is based on the spectral signal-to-noise ratio and resolution. To distinguish between intra- and intermolecular long-range contacts, mixed labeled and diluted samples have revealed efficient.
In short, the critical steps for an atomic SSNMR structural study are: (i) the preparation of the subunits and the assembly need to be optimized to obtain excellent sample quantity and quality, (ii) spectrometer field strength and acquisition parameters have to be chosen carefully; (iii) selective labeling strategies are required for a 3D structure determination and the amount of required data depends on data quality and the availability of complementary data.
Despite its applicability to a wide range of supramolecular systems ranging from membrane proteins to homomultimeric nano-objects, SSNMR is often limited by the need for mg-quantities of isotopically labeled material. The recent technological developments in ultra-fast MAS (&#8805;100 kHz) SSNMR open up the avenue to 1H-detected NMR, and push the limit of minimal sample quantity to sub-mg 72,73,74. Nevertheless, for detailed structural studies 13C-labeled samples are indispensable, which limits the application of SSNMR to samples assembled in vitro or to systems expressed in organisms that survive on minimal medium where in-cell SSNMR is an emerging method (for reviews see 75,76,77,78).
An important factor in SSNMR application to obtain high-resolution 3D structures is the spectral resolution: intrinsic conformational heterogeneity in an assembly can limit spectral resolution and spectra analysis. Residue specific 13C labeling may in some cases provide an alternative to obtain specific distance information on strategic residues in order to obtain structural models (for a recent examples see 79,80).
SSNMR for 3D structure determination still requires the collection of several datasets with often long data collection times on sophisticated instruments, depending on the approach and the system several days to weeks on a 600-1000 MHz (1H frequency) spectrometer. Therefore, the access to spectrometer time can be a limiting factor in an in-depth SSNMR study.
In the case of homomultimeric protein assemblies, leading to SSNMR data of sufficient quality to identify a high number of structural restraints such as in 3,57,64,70, SSNMR still gives no access to the microscopic dimensions. Therefore, in a de novo SSNMR structure determination of a homomultimeric assembly, EM or mass-per-length (MPL) data ideally complement SSNMR data to derive the symmetry parameters. SSNMR data alone provide the atomic intra- and intermolecular interfaces
SSNMR is highly complementary with structural techniques such as EM or MPL measurements but the data can also perfectly be combined with atomic structures obtained by X-ray crystallography or solution NMR on mutated or truncated subunits. An increasing number of studies can be found in literature where the conjunction of different structural data has allowed for determining atomic 3D models of macromolecular assemblies (see Figure 6 for representative examples).
In the field of Structural Biology, SSNMR emerges as promising technique to study insoluble and non-crystalline assemblies at the atomic level, i.e. providing structural data at the atomic scale. In this respect, SSNMR is the pendant to solution NMR and X-ray crystallography for molecular assemblies, including membrane proteins in their native environment and protein assemblies such as viral envelopes, bacterial filaments or amyloids, as well as RNA and RNA-protein complexes (see for example81). Its highly versatile applications in vitro and in the cellular context, such as tracking secondary, tertiary and quaternary structural changes, identifying interaction surfaces with partner molecules on the atomic scale (for example 82) and mapping molecular dynamics in the context of assembled complexes, indicate the important potential of SSNMR in future structural studies on complex biomolecular assemblies.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>M9 medium</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>0.5 g/L</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>3 g/L</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>6.7 g/L</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>10 &#956;M</td>
		</tr>
		<tr>
			<td>FeCl3</td>
			<td>1 &#956;M</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>MEM vitamin mix 100X</td>
			<td>10 mL/L</td>
		</tr>
		<tr>
			<td>13C-glucose</td>
			<td>2 g/L</td>
		</tr>
		<tr>
			<td>15NH4Cl</td>
			<td>1 g/L</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of minimal expression medium for recombinant protein production in E. coli BL21 cells.

Advances in magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (SSNMR) offer an efficient tool for the structural characterization of macromolecular protein assemblies at an atomic resolution. These protein assemblies are ubiquitous systems that play essential roles in many biological processes. Their molecular structures, interactions and dynamics are accessible by SSNMR studies, as has been shown for viral (capsids1) and bacterial infection mechanisms (secretion systems2,3, pili4), membrane protein complexes5,6,7,8 and functional amyloids 9,10,11. This type of molecular assembly can also provoke pathologies such as in neurodegenerative diseases where proteins assemble in misfolded, amyloid states and cause aberrant cell behavior or cell death 12,13. Protein assemblies are often built by the symmetric oligomerization of multiples copies of protein subunits into large supramolecular objects of various shapes including fibrils, filaments, pores, tubes, or nanoparticles. The quaternary architecture is defined by weak interactions between protein subunits to organize the spatial and temporal assembly and to allow for sophisticated biological functions. Structural investigations at an atomic scale on these assemblies are a challenge for high-resolution techniques since their intrinsic insolubility and very often their non-crystallinity restricts the use of conventional X-ray crystallography or solution NMR approaches. Magic-angle spinning (MAS) SSNMR is an emerging technique to obtain atomic resolution data on insoluble macromolecular assemblies and has proven its efficiency to resolve 3D atomic models for an increasing number of complex biomolecular systems including bacterial filaments, amyloid assemblies and viral particles 14,15,16,17,18,19,20,21,22. Technical advances on high magnetic fields, methodological developments and sample preparation has established MAS SSNMR into a robust method to investigate insoluble proteins in various environments, notably in their biologically-relevant macromolecular assembled state or in cellular membranes, making the technique highly complementary to cryo-electron microscopy. In many cases, a very high degree of symmetry characterizes such protein assemblies. MAS SSNMR exploits this feature, as all protein subunits in a homomolecular assembly would have the same local structure and therefore virtually the same SSNMR signature, drastically reducing the complexity of the analysis.
An efficient protocol for structural studies of macromolecular protein assemblies by moderate MAS (&#60;25 kHz) SSNMR is presented in this video and can be subdivided into different steps (Figure 1). We will demonstrate the critical stages of the workflow of a SSNMR structural study exemplified on a filamentous protein assembly (see highlighted steps in Figure 1), with the exception of protein subunit purification, differing for each protein assembly but of critical importance for structural studies, and without going into the technical/methodological details of SSNMR spectroscopy and structure calculation for what specialized tutorials are available online. While the present protocol will primarily focus on solid-state NMR experiments performed under MAS conditions, the use of aligned biological environments 23,24,25,26,27, such as aligned bicelles, allow for the investigation of protein conformation and dynamic protein-protein interaction in membrane-like media without MAS technology. We will show the protein expression and assembly steps as well as the recording of the crucial SSNMR spectra and their analysis and interpretation. Our aim is to provide insights into the structural analysis pipeline enabling the reader to perform an atomic-resolution structural study of a macromolecular assembly by SSNMR techniques.
The protocol encompasses 3 sections:
1. Solid-state NMR sample production
As a prerequisite to a solid-state NMR analysis, the protein components of the macromolecular assembly need to be expressed, isotope-labeled, purified and assembled in vitro into the native-like complex state (for an example see Figure 2). To ensure high NMR sensitivity, isotope enrichment in 13C and 15N labeling is required through the use of minimal bacterial expression media supplemented with 13C and 15N sources, such as uniformly 13C-labeled glucose/glycerol and 15NH4Cl respectively. In the later stage of the protocol, selectively 13C-labeled samples produced with selectively 13C-labeled sources such as (1,3-13C)- and (2-13C)-glycerol (or (1-13C)- and (2-13C)-glucose) are used to facilitate the NMR analysis. Mixed labeled sample corresponding to an equimolar mixture of either 50% 15N- and 50% 13C-labeled or 50% (1,3-13C)- and 50% (2-13C)-glucose are introduced to describe the detection of intermolecular interactions. A high degree of protein purity as well as rigorous conditions during the assembly step are key factors to insure a homogeneous structural order of the final sample.
2. Preliminary structural characterization based on one-dimensional (1D) solid-state NMR
We present the essential experiments for a structural analysis by SSNMR. One-dimensional (1D) cross-polarization (CP) and INEPT / RINEPT28 experiments, detected on 13C nuclei are used to detect rigid and flexible protein segments in the assembly, respectively, and to estimate the degree of structural homogeneity and local polymorphism (for an example see Figure 3).
3. Conformational analysis and 3D structure determination
Subsections 1 and 2 concern the conformational analysis, which is based on the SSNMR resonance assignment of all rigid residues of the protein assembly, as the chemical shifts are very sensitive probes to the local environment and can be used to predict the phi/psi dihedral angles and thereby determine the secondary structure. Figure 4 illustrates an example of a sequential resonance assignment in the rigid core of a protein assembly. The 3D structure determination is based on the collection of structural data such as distance restraints encoding close proximities (&#60;7 - 9 &#197;), containing both intra- and intermolecular information. Subsections 3 and 4 describe long-range distant restraint collection and interpretation. Long-range contacts are defined as intramolecular 13C-13C proximities arising from residue i to j, with |i-j| &#8805;4, defining thereby the tertiary protein fold of the monomeric subunit, or as intermolecular 13C-13C proximities, defining the intermolecular interfaces between protein subunits in the assembly. Intra- and intermolecular interfaces are illustrated in Figure 5. SSNMR restraints detected through 13C-13C and 15N-13C recoupling experiments usually encode for internuclear distances &#60; 1 nm. Subsection 4 explains the detection of intermolecular distance restraints. In symmetrical protein assemblies, the use of homogeneously labeled samples (i.e. 100% uniformly or selectively labeled) for identifying intermolecular subunit-subunit interactions is limited, as both intra- and inter-molecular contacts lead to detectable signals. The unambiguous detection of intermolecular proximities is achieved by using mixed labeled samples, containing an equimolar mixture of two differently labeled samples, combined prior to aggregation. Subsection 5 briefly introduces structure modeling.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55779/55779fig1.jpg" /> 
Figure 1:&#160;Workflow of an atomic-resolution structural study by solid-state NMR.13C,&#160;15N isotope labeled protein production, subunit purification, subunit assembly, control of assembly formation, SSNMR experiments, SSNMR experiment analysis and extraction of distance restraints, and structure modeling are shown.&#160;<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55779/55779fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NMR Spectrometer (&gt; 11.7 Tesla)</td><td>Bruker</td><td>-</td><td></td></tr><tr><td>triple resonance MAS SSNMR probehead&amp;nbsp;</td><td>Bruker</td><td>-</td><td></td></tr><tr><td>SSNMR rotors 4mm</td><td>Bruker</td><td>K1910</td><td></td></tr><tr><td>Centrifuge 5804 R&amp;nbsp;</td><td>Eppendorf</td><td>5805000629</td><td></td></tr><tr><td>GeneQuant 1300 spectrometer</td><td>Dutscher</td><td>28-9182-13</td><td></td></tr><tr><td>IGS60 INCUBATEUR HERATHERM 75 L</td><td>Dutscher</td><td>228001</td><td></td></tr><tr><td>MaxQ 4450 bench top orbital shaker</td><td>Dutscher</td><td>78376</td><td></td></tr><tr><td>Tube Revolver Agitator</td><td>Dutscher</td><td>79547</td><td></td></tr><tr><td>sonopuls HD 3100</td><td>Bandelin</td><td>3680</td><td></td></tr><tr><td>MicroPulser electroporator</td><td>Biorad</td><td>165-2100&amp;nbsp;</td><td></td></tr><tr><td>mini-PROTEAN tetra cell system</td><td>Biorad</td><td>165-8000</td><td></td></tr><tr><td>AKTA pure system</td><td>GE Healthcare</td><td>29-0182-24</td><td></td></tr><tr><td>capillary microman M25 pipet</td><td>Gilson&amp;nbsp;</td><td>F148502</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>amicon&lt;sup&gt;R&lt;/sup&gt; ultra-15</td><td>sigma</td><td>Z740199-8EA</td><td></td></tr><tr><td>capillaries and pistons</td><td>Gilson&amp;nbsp;</td><td>F148112</td><td></td></tr><tr><td>spatula</td><td>Fisher</td><td>13263799</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D-glucose &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>389374</td><td></td></tr><tr><td>Ammonium-15N-chloride</td><td>Sigma</td><td>299251</td><td></td></tr><tr><td>1,3 &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;2&lt;/sub&gt; glycerol</td><td>Sigma</td><td>492639</td><td></td></tr><tr><td>2 &lt;sup&gt;13&lt;/sup&gt;C glycerol</td><td>Sigma</td><td>489484</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma</td><td>K1876</td><td></td></tr><tr><td>Carbenicillin</td><td>Sigma</td><td>C3416</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>71380</td><td></td></tr><tr><td>calcium chloride</td><td>Sigma</td><td>C1016</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>208094</td><td></td></tr><tr><td>Iron Chloride</td><td>Sigma</td><td>157740</td><td></td></tr><tr><td>Zinc chloride</td><td>Sigma</td><td>793523</td><td></td></tr><tr><td>MEM Vitamin Solution (100&amp;times;)</td><td>Sigma</td><td>M68954</td><td></td></tr><tr><td>IPTG</td><td>Fisher</td><td>BP1755</td><td></td></tr><tr><td>Trizma base&amp;nbsp;</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td>436143</td><td></td></tr><tr><td>sodium azide</td><td>sigma</td><td>71289</td><td></td></tr><tr><td>4,4-dimethyl-4-silapentane-1-sulfonic acid&amp;nbsp;</td><td>Sigma</td><td>178837</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Unicorn 6.3</td><td>GE Healthcare</td><td></td><td>Akta systems</td></tr><tr><td>ccpNMR</td><td>CCPN&amp;nbsp;</td><td></td><td>spectrometer systems</td></tr></tbody></table>

atomic scale structural studies of macromolecular assemblies by solid-state nuclear magnetic resonance spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

The typical SSNMR workflow includes several steps illustrated in Figure 1. Usually the protein subunits are produced by in vitro heterologous expression in E. coli, purified and assembled under shaking but sometimes also in static conditions. Expression and purification of the protein subunit are followed by SDS gel chromatography (Figure 2A). The formation of macromolecular assemblies can then be confirmed by electron microscopy (EM) analysis (see Figure 2B for an example of a filamentous assembly).
After introduction of the protein assembly into the SSNMR rotor, the rotor is inserted into the spectrometer, the MAS frequency and temperature are regulated and the spectra are recorded. First insights can be obtained by 1D SSNMR techniques. Figure 3 shows a typical SSNMR FID detected on the 13C channel on a structurally homogeneous protein sample, a 1H-13C CP spectrum, revealing the13C resonances present in the rigid core of the protein subunit in the assembly, and a 2D 1H-13C INEPT spectrum, representing the mobile residues. For atomic insights into the rigid core of the assembly structure, multidimensional SSNMR experiments need to be recorded on uniformly and selectively labeled samples to first assign the SSNMR resonances and then detect long-range proximities (see Figure 4).
All spectra are processed and analyzed with adequate software to assign the SSNMR resonances and extract intra- and intermolecular distance restraints (Figure 5). The SSNMR distance restraints are either used alone or in conjunction with data from complementary techniques, which can be integrated into the modeling program.
For representative atomic structures of macromolecular assemblies solved by SSNMR techniques Figure 6 illustrates several filamentous assemblies from bacterial appendages and amyloid fibrils.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55779/55779fig6v2.jpg" /> 
Figure 6:Filamentous macromolecular structures determined by a solid-state NMR approach: bacterial filaments and amyloid protein fibrils.A) Type 1 pilus of uropathogenic E. coli, PDB code 2N7H 4; B) ASC filament, PDB code 2N1F 63; C) Type III secretion system needles, PDB codes 2MME, 2LPZ and 2MEX 2,3,64; D) Amyloid-beta AB42 fibrils, PDB code 2NAO, 5KK3, 2MXU 65,66,67 and Osaka mutant PDB code 2MVX 57, Iowa mutant PDB code 2MPZ 58; E) Alpha-synuclein fibrils, PDB code 2N0A 68; F) HET-s prion domain, PDB code 2RNM, 2KJ3 69,70. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55779/55779fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

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15.4K Views.  UMR5248 CNRS, Université de Bordeaux. Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Video: Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic glasses (MGs). Despite their amorphous nature, the identification of hyperfine interactions unveils faint structural modifications. For this purpose, we have employed two techniques that utilize nuclear resonance among nuclear levels of a stable 57Fe isotope, namely M&#246;ssbauer spectrometry and nuclear forward scattering (NFS) of synchrotron radiation. The effects of heat treatment upon (Fe2.85Co1)77Mo8Cu1B14 MG are discussed using the results of ex situ and in situ experiments, respectively. As both methods are sensitive to hyperfine interactions, information on structural arrangement as well as on magnetic microstructure is readily available. M&#246;ssbauer spectrometry performed ex situ describes how the structural arrangement and magnetic microstructure appears at room temperature after the annealing under certain conditions (temperature, time), and thus this technique inspects steady states. On the other hand, NFS data are recorded in situ during dynamically changing temperature and NFS examines transient states. The use of both techniques provides complementary information. In general, they can be applied to any suitable system in which it is important to know its steady state but also transient states.

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

Here, we present a protocol to describe ex situ and in situ investigations of structural transformations in metallic glasses. We employed nuclear-based analytical methods which inspect hyperfine interactions. We demonstrate the applicability of M&#246;ssbauer spectrometry and nuclear forward scattering of synchrotron radiation during temperature-driven experiments.

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic ...

Engineering

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There exists a drive to characterize materials under conditions relevant to the chemical process of interest. To address this, in situ high-temperature, high-pressure MAS NMR methods have been developed to enable the observation of chemical interactions over a range of pressures (vacuum to several hundred bar) and temperatures (well below 0 &#176;C to 250 &#176;C). Further, the chemical identity of the samples can be comprised of solids, liquids, and gases or mixtures of the three. The method incorporates all-zirconia NMR rotors (sample holder for MAS NMR) which can be sealed using a threaded cap to compress an O-ring. This rotor exhibits great chemical resistance, temperature compatibility, low NMR background, and can withstand high pressures. These combined factors enable it to be utilized in a wide range of system combinations, which in turn permit its use in diverse fields as carbon sequestration, catalysis, material science, geochemistry, and biology. The flexibility of this technique makes it an attractive option for scientists from numerous disciplines.

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There ...

Chemistry

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Protocol for NMR 15N Relaxation and hetNOE Experiments in Protein Dynamics Study

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

Author Spotlight: Exploring Intrinsically Disordered Protein Dynamics Through NMR Relaxation Experiments

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

Nuclear Magnetic Resonance (NMR) Spectroscopy

Source: Laboratory of Dr. Henrik Sund&#233;n &#8211; Chalmers University of Technology

Nuclear magnetic resonance (NMR) spectroscopy is a vital analysis technique for organic chemists. With the help of NMR, the work in the organic lab has been facilitated tremendously. Not only can it provide information about the structure of a molecule but also determine the content and purity of a sample. Compared with other commonly encountered techniques for organic chemists &#8212; such as thermal analysis and mass spectrometry (MS) &#8212; NMR is a non-destructive method that is valuable when recovery of the sample is important.
One of the most frequently used NMR techniques for an organic chemist is proton (1H) NMR. The protons present in a molecule will behave differently depending on its surrounding chemical environment, making it possible to elucidate its structure. Moreover, it is possible to monitor the completion of a reaction by comparing NMR spectra of the starting material to that of the final product.
This video exemplifies how NMR spectroscopy can be used in the everyday work of an organic chemist. The following will be shown: i) preparation of an NMR sample. ii) Using 1H NMR to monitor a reaction. iii) Identifying the product obtained from a reaction with 1H NMR. The reaction that will be shown is the synthesis of an E-chalcone (3) from an aldehyde (1) and a ketone (2) (Scheme 1).1
<img alt="Scheme 1" src="/files/ftp_upload/5680/5680scheme1.jpg" /> 
Scheme 1. Synthesis of (2E)-3-(4-methoxyphenyl)-1-(4-methylphenyl)-2-propen-1-one.

NMR Spectroscopy: Principle, NMR Active Nuclei, Applications

Source: Laboratory of Dr. Henrik Sund&#233;n &#8211; Chalmers University of Technology

Nuclear magnetic resonance (NMR) spectroscopy is a vital analysis ...

JoVE Science Education

Organic Chemistry

Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Turn on the airflow with the eject command &#34;ej&#34;. This will bring the sample up from the magnet. Now, place the sample within a spinner on top of the magnet in the opening. Insert with the command &#34;ij&#34;. Wait until the sample settles inside the magnet before proceeding.
Create a new data set using the &#34;edc&#34; command, and load Standard Proton NMR parameters by selecting the experiment &#34;ZGPR&#34;. Fill in the name, experiment number, and processed data folder number fields.
Select the solvent in the &#34;Set Solvent&#34; field and click on &#34;Execute &#39;getprosol&#39;&#34; to read standard probehead head and solvent-dependent parameters. Lock the sample to the deuterated solvent using the &#34;Lock&#34; command, and wait until it is finished sweeping and achieves lock.
Correct the resonance frequency of the magnet by tuning the sample using the automatic tuning command &#34;atma&#34;. Monitor the wobble curve until the automatic tuning is complete.
Shim the magnetic field using &#34;TOPSHIM&#34;. Shimming makes adjustment to the magnetic field to achieve uniformity around the sample. It is good practice to store the same values with the command &#34;wsh&#34; and read them using &#34;rsh&#34; before topshim if using the same or similar samples.
Now, adjust the receiver gain with the &#34;rga&#34; command to achieve the maximum signal-to-noise ratio. Place the center of the spectrum on the water resonance offset and set the 90-degree proton pulse at high power using &#34;calibo1p1&#34;.
Collect the proton spectrum using the Zero Go &#34;zg&#34; command and process with &#34;efp&#34;. This includes exponential multiplication, the free induction decay incorporating line broadening, Fourier transformation of FID, and &#34;pk&#34; to apply phase correction.
Apply the automatic phase correction &#34;apk&#34; and the automatic baseline correction &#34;absn&#34; using the polynomial without integration option. Create a new data set for the SOFAST HMBC experiment by selecting &#34;SFHMQC3GPPH&#34; in experiment.
Copy the optimized P1 and O1 from the proton spectrum and populate P1-dependent pulses by using the command &#34;getprosol 1H p1 plw1&#34; where P1 is the optimized P1 value and plw1 is the power level for P1. Now, optimize the CNST54 constant to set the offset for amide chemical shift. Also, optimize CNST55 to define the bandwidth in order to encompass the spectral regions of interest, allowing the receiver gain to be optimized.
To select these parameters, extract the first FID from the two-dimensional spectrum, and look for the observed signal to define them. In addition, vary the relaxation delay, number of scans, and dummy scans to obtain acceptable signal sensitivity with the command &#34;gs&#34;, which enables go and scan to monitor data quality in real-time. Finally, record the spectra using Zero Go &#34;zg&#34;.

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

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Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

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High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

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Nuclear Magnetic Resonance (NMR) Spectroscopy

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