Structure of <i>In Vitro</i>-Synthesized Cellulose Fibrils Viewed by Cryo-Electron Tomography and ¹³C Natural-Abundance Dynamic Nuclear Polarization Solid-State NMR

Abstract

Cellulose, the most abundant biopolymer, is a central source for renewable energy and functionalized materials. <i>In vitro</i> synthesis of cellulose microfibrils (CMFs) has become possible using purified cellulose synthase (CESA) isoforms from <i>Physcomitrium patens</i> and hybrid aspen. The exact nature of these <i>in vitro</i> fibrils remains unknown. Here, we characterize <i>in vitro</i>-synthesized fibers made by CESAs present in membrane fractions of <i>P. patens</i> over-expressing CESA5 by cryo-electron tomography and dynamic nuclear polarization (DNP) solid-state NMR. DNP enabled measuring two-dimensional ¹³C-¹³C correlation spectra without isotope-labeling of the fibers. Results show structural similarity between <i>in vitro</i> fibrils and native CMF in plant cell walls. Intensity quantifications agree with the 18-chain structural model for plant CMF and indicate limited fibrillar bundling. The <i>in vitro</i> system thus reveals insights into cell wall synthesis and may contribute to novel cellulosic materials. The integrated DNP and cryo-electron tomography methods are also applicable to structural studies of other carbohydrate-based biomaterials.