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

This protocol can be used to prepare fungal and plant materials for solid-state nuclear magnetic resonance, or NMR, and dynamic nuclear polarization, or DNP, experiments for complex biosystem characterization. This technique allows us to investigate biomaterials to an atomic resolution level in their native environment, for example, in whole cells. The acquisition of high-resolution structural information about fungal materials will aid in development of antifungal drugs.

The method provides insight into composition and structure of complex carbohydrates in fungal cell walls and can be applied to many carbohydrate-rich organisms, including plants, fungi, algae, and bacteria. Some investigators may struggle with fungal contamination in their cellular cultures. My advice is to sterilize the medium and equipment thoroughly before use to prevent this contamination.

This demonstration will help investigators with little experience in NMR or cell culture to learn how to implement these techniques in fungal and plant systems. To prepare unlabeled liquid growth medium, dissolve 6.5 grams of yeast extract peptone dextrose or YPD powder in 100 milliliters of distilled water, and then autoclave the resulting solution for 25 minutes at 134 degrees Celsius. To prepare unlabeled solid growth medium, add 6.5 grams of YPD agar powder to 100 milliliters of distilled water, and autoclave the medium for 25 minutes at 121 degrees Celsius, before cooling the medium to approximately 50 degrees Celsius.

Then, transfer 13 to 15 milliliters of the melted solid growth medium into individual sterile plastic Petri dishes, and immediately place the lids onto the dishes. To prepare carbon-13, nitrogen-15-labeled liquid growth medium, adjust a 100-milliliter volume of isotope-containing minimal medium to a pH of 6.6 with acid or base as necessary. Next, dissolve the salts listed in the table in 100 milliliters of distilled water, add to the carbon-13, nitrogen-15-labeled liquid growth medium, and autoclave the resulting solution for 25 minutes at 121 degrees Celsius.

When the solution has cooled to room temperature, add 100 microliters of trace elements solution to the entire volume of carbon-13, nitrogen-15-labeled minimal medium. To grow the fungal materials, use an inoculating loop to transfer a small amount of fungi from storage onto a YPD plate, and culture the fungus for two days in a 30-degree Celsius incubator. At the end of the incubation, use a new inoculating loop to transfer an active growing fungal edge to the carbon-13, nitrogen-15-labeled liquid growth medium, and place the culture at 30 degrees Celsius and 220 rotations per minute for three to five days in a shaking incubator.

When large quantities of the fungus cover the flask bottom and float in the liquid, collect the fungi by centrifugation and discard the supernatant. Hydrate the pellet with an appropriate solution, and use forceps to collect about 0.5 grams of the well-hydrated pellet for NMR and DNP analyses. Then, mix the remaining material with a 20%glycerol solution in a conical tube, and place the fungal sample at minus 80 degrees Celsius for long-term storage.

To prepare the A.fumigatus for solid-state NMR analysis, first use a dialysis bag with a 3.5-kilodalton molecular weight cutoff to dialyze the 0.5-gram carbon-13, nitrogen-15-labeled fungal sample against one liter of 10-millimolar phosphate buffer at four degrees Celsius for three days to remove small molecules from the growth medium. At the end of the dialysis, transfer the sample into a 15-milliliter tube for centrifugation, and pack 70 to 80 milligrams of the uniformly carbon-13-labeled and well-hydrated sample paste into a four-millimeter zirconium dioxide rotor. Use a metal rod to gently and repetitively squeeze the sample, using paper to absorb the excess water.

Then, tightly cap the rotor, and insert the sample into the spectrometer for solid-state NMR characterization. To prepare the A.fumigatus for DNP analysis, add 100 microliters of DNP matrix into one 1.5-milliliter microcentrifuge tube per carbon-13, carbon-15-labeled fungal sample, and dissolve 0.7 milligrams of polarizing agent to each tube to form a 10-millimolar radical stock solution. After vortexing for two to three minutes to ensure that radicals are fully dissolved in the solution, soak 10 milligrams of dialyzed carbon-13, nitrogen-15-labeled fungal sample in 50 microliters of the polarizing agent solution, and use a pestle and a mortar to mildly grind the mixture to ensure penetration of the radicals into the porous cell walls.

Add another 30 microliters of the radical solution to the ground pellet to further hydrate the fungal sample, and pack the pellet into a 3.2-millimeter sapphire rotor. Squeeze mildly and remove the excess DNP solvent as demonstrated, and add a 3.2-millimeter silicone plug to prevent the loss of hydration. Then, add the rotor to an NMR spectrometer for routine experiment or a DNP spectrometer for measurement of the DNP-enhanced spectrum under microwave irradiation.

To prepare plant materials for DNP studies, use a razor blade to cut the uniformly carbon-13-labeled plant material into one-to two-millimeter pieces. Use a mortar and pestle to grind the pieces into smaller particles until the final powder has a homogenous appearance. Add 40 microliters of 10-millimolar radical stock solution to the plant material, and lightly grind the sample for an additional five minutes to ensure homogeneous mixing with the radical.

Hydrate the ground sample with another 20 microliters of radical stock solution, and pack the equilibrated plant sample into a 3.2-millimeter sapphire rotor for DNP analysis. Then, insert a silicone plug to avoid the loss of hydration, and load the sample onto the DNP spectrophotometer. Isotope labeling substantially enhances the NMR sensitivity and makes it possible for measuring a series of two-dimensional carbon-13-carbon-13 and carbon-13-nitrogen-15 correlation spectra to analyze the composition, hydration, mobility, and packing of the polymers for their integration for construction of a three-dimensional model of cell wall architecture.

If off-diagonal signals are difficult to obtain in the 2D carbon-13-carbon-13 spectrum, statistical labeling might have occurred. The two carbon-13 peaks at 96 and 92 parts per million are signature carbon-1 signals of glucose. Therefore, their strong intensities in the quantitative carbon-13 direct polarization spectra measured with long recycle delays of 35 seconds typically indicate the dominance of small molecules due to incomplete dialysis or washing.

With well-labeled samples, long-range correlations can be further measured to detect the spatial proximities of biomolecules and to construct a structural model of the intact cell walls. This technique allows structure function in many natural-occurring and engineered materials to be explored, facilitating future studies of carbohydrate-rich biomaterials and functionalized polymers. As some fungi may cause infection or systematic diseases in humans, be sure to handle fungal materials in a laminar flow hood for protection and to minimize exposure.