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

Summary

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.

Abstract

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.

Introduction

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 bacteria^1,2,3. The cell wall serves as a central source for the production of biofuel and biomaterials, as well as a promising target for antimicrobial therapies^4,5,6,7,8<....

Protocol

1. Growth of ¹³C, ¹⁵N-labeled Aspergillus fumigatus Liquid Medium

1. 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.
   1. Preparation of unlabeled liquid medium: Dissolve 6.5 g of YPD powder in 100 mL of distilled water and then autoclave for 25 min at 134 °C.
   2. Preparation of unlabeled solid medium
      1. Add 1....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

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