Solid-state nmr and dnp investigations of carbohydrates and cell-wall biomaterials

© 2020 John Wiley & Sons, Ltd. The cell walls in plants and microbes serve as a central source for biorenewable energy and biomaterials, as well as the target for novel antibiotics and antifungals. They are biocomposites abundant in complex carbohydrates, a class of biologically important but underinvestigated molecules. Solid-state nuclear magnetic resonance (ssNMR) of carbohydrate materials and cell walls has made significant progress over the past 10 years. This article summarizes the recent ssNMR studies that have elucidated the polymorphic structure and heterogeneous dynamics of polysaccharides and other biomolecules, such as proteins, lignin, and pigment, in the intact cell walls or biofilms of 11 species across plants, fungi, bacteria, and algae. We also highlight the assistance of magic-angle spinning dynamic nuclear polarization (MAS-DNP) in the enhanced detection of the interaction interface involving lowly populated biopolymers and summarize the recent applications of natural-abundance MAS-DNP in cell-wall research, which could substantially broaden the scope of biomolecular NMR by skipping isotope labeling

CCMRD: a solid-state NMR database for complex carbohydrates

© 2020, Springer Nature B.V. Carbohydrates are essential to various life activities in living organisms and serve as the central component in many biomaterials. As an emerging technique with steadily improving resolution, solid-state Nuclear Magnetic Resonance (NMR) spectroscopy has the unique capability in revealing the polymorphic structure and heterogeneous dynamics of insoluble complex carbohydrates. Here, we report the first solid-state NMR database for complex carbohydrates, Complex Carbohydrates Magnetic Resonance Database (CCMRD). This database currently holds the chemical shift information of more than four hundred solid-state NMR compounds and expects rapid expansion. CCMRD provides open portals for data deposition and supports search options based on NMR chemical shifts, carbohydrate names, and compound classes. With the timely implementation, this platform will facilitate spectral analysis and structure determination of carbohydrates and promote software development to benefit the research community. The database is freely accessible at www.ccmrd.org

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

Atomic resolution of cotton cellulose structure enabled by dynamic nuclear polarization solid-state NMR

© 2018, Springer Nature B.V. Abstract: The insufficient resolution of conventional methods has long limited the structural elucidation of cellulose and its derivatives, especially for those with relatively low crystallinities or in native cell walls. Recent 2D/3D solid-state NMR studies of 13 C uniformly labeled plant biomaterials have initiated a re-investigation of our existing knowledge in cellulose structure and its interactions with matrix polymers but for unlabeled materials, this spectroscopic method becomes impractical due to limitations in sensitivity. Here, we investigate the molecular structure of unlabeled cotton cellulose by combining natural abundance 13 C– 13 C 2D correlation solid-state NMR spectroscopy, as enabled by the sensitivity-enhancing technique of dynamic nuclear polarization, with statistical analysis of the observed and literature-reported chemical shifts. The atomic resolution allows us to monitor the loss of Iα and Iβ allomorphs and the generation of a novel structure during ball-milling, which reveals the importance of large crystallite size for maintaining the Iα and Iβ model structures. Partial order has been identified in the “disordered” domains, as evidenced by a discrete distribution of well-resolved peaks. This study not only provides heretofore unavailable high-resolution insights into cotton cellulose but also presents a widely applicable strategy for analyzing the structure of cellulose-rich materials without isotope-labeling. This work was part of a multi-technique study of ball-milled cotton described in the previous article in the same issue. Graphical abstract: [Figure not available: see fulltext.]

Lignin-polysaccharide interactions in plant secondary cell walls revealed by solid-state NMR

© 2019, The Author(s). Lignin is a complex aromatic biopolymer that strengthens and waterproofs plant secondary cell walls, enabling mechanical stability in trees and long-distance water transport in xylem. Lignin removal is a key step in paper production and biomass conversion to biofuels, motivating efforts to re-engineer lignin biosynthesis. However, the physical nature of lignin’s interactions with wall polysaccharides is not well understood. Here we show that lignin self-aggregates to form highly hydrophobic and dynamically unique nanodomains, with extensive surface contacts to xylan. Solid-state NMR spectroscopy of intact maize stems, supported by dynamic nuclear polarization, reveals that lignin has abundant electrostatic interactions with the polar motifs of xylan. Lignin preferentially binds xylans with 3-fold or distorted 2-fold helical screw conformations, indicative of xylans not closely associated with cellulose. These findings advance our knowledge of the molecular-level organization of lignocellulosic biomass, providing the structural foundation for optimization of post-harvest processing for biofuels and biomaterials

Structural adaptation of fungal cell wall in hypersaline environment

Abstract Halophilic fungi thrive in hypersaline habitats and face a range of extreme conditions. These fungal species have gained considerable attention due to their potential applications in harsh industrial processes, such as bioremediation and fermentation under unfavorable conditions of hypersalinity, low water activity, and extreme pH. However, the role of the cell wall in surviving these environmental conditions remains unclear. Here we employ solid-state NMR spectroscopy to compare the cell wall architecture of Aspergillus sydowii across salinity gradients. Analyses of intact cells reveal that A. sydowii cell walls contain a rigid core comprising chitin, β-glucan, and chitosan, shielded by a surface shell composed of galactomannan and galactosaminogalactan. When exposed to hypersaline conditions, A. sydowii enhances chitin biosynthesis and incorporates α-glucan to create thick, stiff, and hydrophobic cell walls. Such structural rearrangements enable the fungus to adapt to both hypersaline and salt-deprived conditions, providing a robust mechanism for withstanding external stress. These molecular principles can aid in the optimization of halophilic strains for biotechnology applications