Name: preparation of fungal and plant materials for structural elucidation using dynamic nuclear polarization solid-state nmr
Uploaded: 2019-02-12T14:30:00
Description: Watch this Scientific Journal Video about Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR at JoVE.com

This work was supported by National Science Foundation through NSF OIA-1833040. The National High Magnetic Field Laboratory (NHMFL) is supported by National Science Foundation through DMR-1157490 and the State of Florida. The MAS-DNP system at NHMFL is funded in part by NIH S10 OD018519 and NSF CHE-1229170.

1. Growth of 13C, 15N-labeled Aspergillus fumigatus Liquid Medium
<ol>
	<li>Preparation of unlabeled and 13C, 15N-labeled growth medium 
	NOTE: Both Yeast Extract Peptone Dextrose medium (YPD) and the improved minimal medium43 were used for the maintenance of fungal culture. All steps after autoclaving are performed in a laminar flow hood to minimize contamination.
	<ol>
		<li>Preparation of unlabeled liquid medium: Dissolve 6.5 g of YPD ...

Compared with the biochemical methods, solid-state NMR has advantages as a non-destructive and high-resolution technique. NMR is also quantitative in compositional analysis, and unlike most other analytical methods, does not have the uncertainties introduced by the limited solubility of biopolymers. Establishment of the current protocol facilitates future studies on carbohydrate-rich biomaterials and functionalized polymers. However, it should be noted that the resonance assignment and data analysis can be time-consuming...

Carbohydrates play a central role in various biological processes such as energy storage, structural building, and cellular recognition and adhesion. They are enriched in the cell wall, which is a fundamental component in plants, fungi, algae and bacteria1,2,3. The cell wall serves as a central source for the production of biofuel and biomaterials, as well as a promising target for antimicrobial therapies4,5,6,7,8,9.
The contemporary understanding of these complex materials has been substantially advanced by decades of efforts that were devoted to the structural characterization using four major biochemical or genetic methods. The first major method relies on sequential treatments using harsh chemicals or enzymes to break down the cell walls into different portions, which is followed by compositional and linkage analysis of sugars in each fraction10. This method sheds light on the domain distribution of polymers, but the interpretation may be misleading due to the chemical and physical properties of biomolecules. For example, it is difficult to determine whether the alkali-extractable fraction originates from a single domain of less structured molecules or from spatially separated molecules with comparable solubility. Second, the extracted portions or whole cell walls can also be measured using solution NMR to determine the covalent linkages, also termed as crosslinking, between different molecules11,12,13,14,15. In this way, the detailed structure of covalent anchors could be probed, but limitations may exist due to the low solubility of polysaccharides, the relatively small number of crosslinking sites, and the ignorance of non-covalent effects that stabilizes polysaccharide packing, including the hydrogen-bonding, van der Waals force, electrostatic interaction and polymer entanglement. Third, the binding affinity has been determined in vitro using isolated polysaccharides16,17,18,19, but the purification procedures may substantially alter the structure and properties of these biomolecules. This method also fails to replicate the sophisticated deposition and assembly of macromolecules after biosynthesis. Finally, the phenotype, cell morphology and mechanical properties of genetic mutants with attenuated production of certain cell wall component shed lights on the structural functions of polysaccharides, but more molecular evidence is needed to bridge these macroscopic observations with the engineered function of protein machineries20.
Recent advances in the development and application of multidimensional solid-state NMR spectroscopy have introduced a unique opportunity for solving these structural puzzles. 2D/3D solid-state NMR experiments enable high-resolution investigation of the composition and architecture of carbohydrate-rich materials in the native state without major perturbation. Structural studies have been successfully conducted on both primary and secondary cell walls of plants, the catalytically treated biomass, bacterial biofilm, the pigment ghosts in fungi and, recently by the authors, the intact cell walls in a pathogenic fungus Aspergillus fumigatus21,22,23,24,25,26,27,28,29,30,31. The development of dynamic nuclear polarization (DNP)32,33,34,35,36,37,38,39,40,41,42 substantially facilitates NMR structural elucidation as the sensitivity enhancement by DNP markedly shortens the experimental time on these complex biomaterials. The protocol described here details the procedures for isotope-labeling the fungus A. fumigatus and preparing fungal and plant samples for solid-state NMR and DNP characterization. Similar labeling procedures should be applicable to other fungi with altered medium, and the sample preparation procedures should be generally applicable to other carbohydrate-rich biomaterials.

<table>
	<tbody>
		<tr>
			<td>Ammonium Molybdate Tetrahydrate</td>
			<td>Acros Organics</td>
			<td>12054-85-2</td>
			<td></td>
		</tr>
		<tr>
			<td>AMUPol</td>
			<td>Cortecnet</td>
			<td>C010P002</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical weighing balance</td>
			<td>Ohaus</td>
			<td>B730439218</td>
			<td>Model PA84C</td>
		</tr>
		<tr>
			<td>Bioclave 16 L</td>
			<td>VWR</td>
			<td>470230-598</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>Labconco corporation</td>
			<td>302319100</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>VWR</td>
			<td>BDH9222</td>
			<td>store at 15-30 &#176;C</td>
		</tr>
		<tr>
			<td>Cobalt(II) Chloride Hexahydrate</td>
			<td>Honeywell|Fluka</td>
			<td>60820</td>
			<td>&#8805;98 %</td>
		</tr>
		<tr>
			<td>Copper(II) Sulfate Pentahydrate</td>
			<td>BDH</td>
			<td>BDH9312</td>
			<td>&#8805;98 %</td>
		</tr>
		<tr>
			<td>Corning LSE shaking incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>7202152</td>
			<td></td>
		</tr>
		<tr>
			<td>D2O</td>
			<td>Sigma Aldrich</td>
			<td>151882</td>
			<td>99.9 atom % D</td>
		</tr>
		<tr>
			<td>d6-DMSO</td>
			<td>Sigma Aldrich</td>
			<td>151874</td>
			<td>99.9 atom % D</td>
		</tr>
		<tr>
			<td>d8-glycerol</td>
			<td>Sigma Aldrich</td>
			<td>447498</td>
			<td>&#8805;99 atom % D</td>
		</tr>
		<tr>
			<td>Dialysis tubing 3.2 kDa</td>
			<td>Sigma Aldrich</td>
			<td>D2272</td>
			<td>132724</td>
		</tr>
		<tr>
			<td>Dipotassium Phosphate</td>
			<td>VWR</td>
			<td>BDH9266</td>
			<td>&#8805;98 %</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>&#8805;99.5 %</td>
		</tr>
		<tr>
			<td>Heraus Megafuge 16R Centrifuge</td>
			<td>Thermo Fischer Scientific</td>
			<td>750004271</td>
			<td>Maximum RCF 25,830 x g</td>
		</tr>
		<tr>
			<td>HR-MAS Disposable Insert Kit</td>
			<td>Bruker</td>
			<td>B4493</td>
			<td>Kel-F</td>
		</tr>
		<tr>
			<td>Iron(II) Sulfate Heptahydrate</td>
			<td>Alfa Aesar</td>
			<td>14498</td>
			<td>&#8805;99+ %</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>VWR</td>
			<td>10034998</td>
			<td>store at 18-26 &#176;C</td>
		</tr>
		<tr>
			<td>Manganese(II) Chloride Tetrahydrate</td>
			<td>Alfa Aesar</td>
			<td>11563</td>
			<td>&#8805;99 %</td>
		</tr>
		<tr>
			<td>Monopotassium Phosphate</td>
			<td>VWR</td>
			<td>470302-254</td>
			<td>&#8805;99 %</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>B706689216</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrasodium Ethylenediaminetetraacetate</td>
			<td>Acros Organics</td>
			<td>13235-36-9</td>
			<td>&#8805;99.5 %</td>
		</tr>
		<tr>
			<td>Zinc Sulfate Heptahydrate</td>
			<td>Alfa Aesar</td>
			<td>33399</td>
			<td>&#8805;98 %</td>
		</tr>
		<tr>
			<td>12C3, d8-glycerol</td>
			<td>Cambridge Isotope Laboratory</td>
			<td>CDLM-8660</td>
			<td>12C3, 99.95%; D8, 98%</td>
		</tr>
		<tr>
			<td>13C6-glucose</td>
			<td>Sigma Alrdrich</td>
			<td>364606</td>
			<td>&#8805;99 % (CP)</td>
		</tr>
		<tr>
			<td>15N-sodium nitrate</td>
			<td>Sigma Aldrich</td>
			<td>364606</td>
			<td>&#8805;98 % 15N, &#8805;99 (cp)</td>
		</tr>
		<tr>
			<td>3.2 mm sapphire NMR rotor</td>
			<td>Cortecnet</td>
			<td>B6939</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm Silicone plug</td>
			<td>Bruker</td>
			<td>B7089</td>
			<td></td>
		</tr>
		<tr>
			<td>4 mm MAS Rotor Kit</td>
			<td>Bruker</td>
			<td>H14355</td>
			<td>Zirconia</td>
		</tr>
	</tbody>
</table>

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.

The isotope labeling substantially enhances the NMR sensitivity and makes it possible for measuring a series of 2D 13C-13C and 13C-15N correlation spectra to analyze the composition, hydration, mobility and packing of polymers, which will be integrated to construct a three-dimensional model of cell wall architecture (Figure 1). If the uniform labeling succeeds, a complete set of 1D 13C and 15N ...

Watch this Scientific Journal Video about Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR at JoVE.com

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

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 ...

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