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^4,5,6,7,8,9.

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 molecules^{11,12,13,14,15}. 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 polysaccharides^16,17,18,19, 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 fumigatus^{21,22,23,24,25,26,27,28,29,30,31}. 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.

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 1.5 g of agar and 6.5 g of YPD powder in 100 mL of distilled water.
      2. Autoclave the medium for 25 min at 121 °C and then cool down to approximately 50 °C.
      3. Transfer 13-15 mL of the medium into each pre-sterile plastic Petri dish and cover the dish using a lid immediately.
   3. Preparation of ¹³C, ¹⁵N-labeled liquid medium
      NOTE: To prepare the growth solution for isotope labeling, a minimal medium containing ¹³C-glucose and ¹⁵N-sodium nitrate and a trace-element solution are prepared separately and then mixed before use.
      1. Prepare 100 mL of the isotope-containing minimal medium as listed in Table 1. Adjust the pH to 6.6 using NaOH (1 M) or HCl (1M) solution.
      2. Autoclave the minimal medium for 25 min at 121 °C.
      3. Prepare 100 mL (1,000x) of trace elements solution, dissolve the salts listed in Table 2 in the distilled water. Autoclave the solution for 25 min at 134 °C. Cool down and store the solution at 4 °C for short-term use. The pH will be about 6.5 and can be checked using a pH meter.
      4. Add 0.1 mL of trace elements solution to 100 mL of ¹³C, ¹⁵N-labeled minimal medium as listed in Table 2 before use.
2. Growth of the fungal materials
   1. Transfer a small amount of fungi from the storage onto a YPD plate using an inoculating loop in a laminar flow hood. Keep the culture at 30 °C for 2 days in an incubator.
   2. Use an inoculating loop to transfer an active growing fungal edge to the ¹³C,¹⁵N-labeling solution in a laminar flow hood. Keep the culture at 30 °C for 3-5 days at 220 rpm in a shaking incubator.
   3. Centrifuge at 4,000 x g for 20 min. Remove the supernatant and collect the pellet.
   4. Use a tweezer to collect ~0.5 g of well-hydrated pellet (>50 wt% hydration) for NMR studies. Loss of hydration at any point will substantially worsen the spectral resolution.
      NOTE: If needed, a small amount (0.1 g) of the hydrated mycelia can be separated and fully dried under N₂ gas flow in a hood or a lyophilizer to estimate the hydration level and calculate the dry mass percentage. Usually, a pellet containing ~0.3 g dry mass can be obtained after 3 days. If the NMR experiment to be conducted is long (>7 days) and/or if the state of the fungi needs to be fixed, the fungal material can be deeply frozen in liquid N₂ for 10-20 min before further processing. If the experiment will be short (3-6 days), the freezing can be skipped so that the sample can remain fresh.
   5. Mix the excess material with 20% (v/v) of glycerol in a centrifuge tube and keep it in a -80 ᵒC freezer for long-term storage.

2. Preparation of A. fumigatus for Solid-state NMR and DNP Studies

1. Preparation of A. fumigatus for solid-state NMR experiments
   1. Dialyze the ¹³C, ¹⁵N-labeled fungal sample (Step 1.2.4.) against 1 L of 10 mM phosphate buffer (pH 7.0) at 4°C using a dialysis bag with a 3.5 kDa molecular weight cutoff to remove small molecules from the growth medium for a total period of 3 days. Change the buffer twice daily.
      NOTE: Alternatively, the sample could be washed for 6-10 times using deionized water to remove residual small molecules.
   2. Transfer the sample into a 15 mL tube and centrifuge for 5 min (10,000 x g) using a benchtop centrifuge. Remove the supernatant and collect the remaining fungal materials.
   3. Pack 70-80 mg of the uniformly ¹³C-labeled and well-hydrated sample paste into a 4-mm ZrO₂ rotor or 30-50 mg to 3.2 mm rotors for NMR experiments. Repetitively squeeze the sample gently using a metal rod and absorb the excess water using paper.
   4. Tightly cap the rotor and insert the sample into the spectrometer for solid-state NMR characterization.
      NOTE: The brand-new rotors are suggested to minimize the possibility of rotor crash and sample spill in the NMR spectrometer. If needed, a disposable Kel-F insert with sealing screws can be used to serve as a secondary container inside the rotor.
2. Preparation of A. fumigatus samples for DNP experiments
   1. Prepare 100 µL of DNP solvents^29,44 (also known as the DNP matrix) in a 1.5 mL microcentrifuge tube for ¹³C,¹⁵N-labeled fungal samples. This DNP matrix contains a mixture of d₈-glycerol/D₂O/H₂O (60/30/10 Vol%).
      NOTE: If unlabeled samples are to be investigated, then prepare the DNP matrix using ¹³C-depleted d₈-glycerol (¹²C₃, 99.95%; D₈, 98%) and D₂O and H₂O to avoid ¹³C signal contribution from the solvents.
   2. Dissolve 0.7 mg of AMUPol⁴⁵ in 100 µL of DNP solvents to form 10 mM radical stock solution. Vortex for 2-3 min to ensure that radicals are fully dissolved in the solution.
   3. Soak 10 mg of the dialyzed ¹³C, ¹⁵N-labeled fungal materials as described in prior steps (Steps 2.1.1 and 2.1.2) into 50 µL of AMUPol solution, and mildly grind the mixture using a pestle and a mortar to ensure penetration of the radicals into the porous cell walls.
      NOTE: To reduce the rate of hydration loss, the grinding can also take place in a microcentrifuge tube using a micropestle.
   4. Add another 30 µL of the radical solution to the grinded pellet to further hydrate the fungal sample.
   5. Pack the pellet into a 3.2-mm sapphire rotor, squeeze mildly and remove the excess DNP solvent. Add a 3.2-mm silicone plug to prevent the loss of hydration. Typically, 5-30 mg of sample can be packed to the rotor. The exact amount needs to be determined by the sensitivity requirement of the NMR experiments to be conducted.
   6. Insert and spin up the sample in a DNP spectrometer, measure a DNP-enhanced spectrum under microwave irradiation and compare it with the microwave-off spectrum. This will lead to an enhancement factor ε_on/off, which should be 20-40 for these complex materials. Run the designed experiments to determine cell wall structure.

3. Preparation of Plant Biomass for NMR and DNP Studies

1. Preparation of plant materials for solid-state NMR
   1. Produce uniformly ¹³C-labeled plants in-house using ¹³CO₂ supplies in a growth chamber or ¹³C-glucose medium as described previously^46,47 or directly purchase labeled materials from isotope-labeling companies.
      NOTE: ¹³C-glucose can only be used in dark growth to avoid the introduction of ¹²C by photosynthesis.
   2. Cut the uniformly ¹³C labeled plant material into small pieces (typically 1-2 mm in dimension) using a laboratory razor blade.
      NOTE: Depending on the purpose, the extracted cell walls are sometimes used for structural characterization and the detailed protocols are reported in previous studies^21,46.
   3. If the sample was previously dried, add 100 µL of water to 30 mg of plant materials in a 1.5 mL microcentrifuge tube, vortex, equilibrate at room temperature for 1 day. Centrifuge at 4,000 x g for 10 min and remove the excess water using a pipette.
   4. If the sample was never-dried at any point, directly use the sample without further treatment.
   5. Pack the resulting plant materials into 3.2-mm or 4-mm ZrO₂ rotors for solid-state NMR experiments.
2. Preparation of plant materials for DNP studies
   1. Prepare 60 µL stock solution of 10 mM AMUPol radical as described steps 2.2.1 and 2.2.2.
   2. Cut the uniformly ¹³C labeled plant material to be studied into small pieces (1-2 mm in dimension) using a laboratory razor blade and weigh 20 mg of the plant materials.
   3. Hand-grind the plant pieces into small particles (~1-2 mm in size) using a mortar and pestle. The final powders have a homogenous appearance.
   4. Add 40 µL of the DNP stock solution prepared in prior step (Step 2.2.2) to the plant material and grind mildly for 5 min to ensure homogeneous mixing with the radical.
   5. Add another 20 µL of the stock solution to further hydrate the plant material after grinding.
   6. Pack the equilibrated plant sample into a 3.2-mm sapphire rotor for DNP experiments. Insert a silicone plug to avoid the loss of hydration.

4. Standard Solid-State NMR Experiments for Initial Characterization of Carbohydrate-Rich Biomaterials

NOTE: A brief overview of the NMR experiments is provided in this section. However, structural elucidation typically requires extensive expertise. Therefore, collaborative efforts with NMR spectroscopists is recommended.

1. Measure 1D ¹³C Cross Polarization (CP), ¹³C Direct Polarization (DP) with 2-s and 35-s recycle delays, and ¹H-¹³C INEPT^48,49 spectra to obtain a general understanding of the dynamical distribution of cell components (Figure 1a). The cell walls are typically the relatively rigid portion and exhibit dominant signals in the CP spectrum.
2. Measure a series of standard 2D ¹³C-¹³C correlation experiments for resonance assignments of ¹³C signals. Start with refocused INADEQUATE^50,51 to obtain carbon connectivity, which need to be assisted by a series of through-space experiments such as 1.5-ms RFDR⁵² (Figure 1b) and 50-ms CORD/DARR⁵³ experiments.
   NOTE: If it is of interest to find a sample rich in a specific component, for example, the primary or secondary cell walls, then multiple segments or multiple plants may need to be measured separately to find the sample with the optimal composition.
3. Conduct 2D ¹⁵N-¹³C correlation experiments which can be measured to facilitate the resonance assignments of proteins and nitrogenated carbohydrates.
   NOTE: The resonance assignment is typically time-consuming. A method is currently being developed to facilitate the resonance assignment of carbohydrate signals for those scientists without prior experience.
4. Measure more specialized experiments to determine the spatial proximities (Figure 1c, d), hydration and mobilities of complex biomolecules to determine the three-dimensional structure of the carbohydrate-rich materials as systematically described previously^22,29.

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