Our overall aim is to further identify, dissect, and exploit dirigent protein (DP) mediated supramolecular protein complexes for macromolecular lignin assembly in order to ultimately achieve facile biomass deconstruction. Our ongoing EMSL work has enabled determination of the structure and oligomerization of the first dirigent (Latin: dirigere to guide or to align) DP. Our proposed study is envisaged to positively impact the EMSL 2019 Science Area Research Focus Topic: "Integrated modeling and structural biology studies to understand enzyme active site chemistry, protein-protein interactions, and other molecular processes of the enzymatic systems involved in biomass deconstruction or metabolic pathways involved in biofuel and bioproduct production".
Lignin/lignocellulosic recalcitrance represent major obstacles in producing cost competitive bioproducts and biofuels at needed scale and cost. This recalcitrance also impacts global carbon recycling (greenhouse gas accumulation and terrestrial carbon turnover in soil and aquatic systems, whether re-mineralized or sequestered). Transformative molecular level discoveries on lignification are thus critical, and DP-mediated processes reportedly generate polymeric lignins, e.g. in Casparian strip (CS) tissue. Here we address organization and function of lignin-forming protein complexes, particularly those providing 1e- oxidation, cofactors and potential scaffolds, in wild type and DP mutant tissues of lignifying Arabidopsis interfascicular fibers, maize pericycle root tissue, and poplar stems. Mutants include "misdirected" lignin deposition in the CS, and secondary (S2 wall layer) depletion with compound middle lamella enriched cell wall stem phenotype.
We request access to 21TFTICR-MS, 750 MHz NMR, and cryo-electron microscopy (cryo-EM) instrumentation, i.e. for establishing the precise nature of DP interacting proteins, as well as for establishing corresponding product molecular structure(s), molecular weights and properties. We will initially need proteomic analyses of the above cell types to identify specific DPs and enzymes in the putative lignin-forming complexes. NanoPOTS (Nanodroplet Processing in One pot for Trace Samples), a new platform for small cell population proteomics analysis developed at PNNL, will be employed.
Overall, we wish to understand and exploit key DP active site chemistries, protein-protein interactions, and redox mediated processes affording specific polymeric lignin entities through tying lignin characterization of LCM micro-dissected tissues together with our studies of DPs and DP-forming complexes. As needed, we will conduct labelling said lignins with 1-13C, 2-13C and 3-13C Phe to facilitate lignin characterization using FTICR-MS and NMR spectroscopy, as well as using stable isotopes to probe further DP structure. We aim to eventually delineate the proteinaceous machinery (protein complex) affording readily cleavable 8-O-4' interunit linked lignin.
Expected results:1) Demonstrate existence of lignin polymerization protein complexes in distinct biomass cell wall types, analogous to CS lignin forming endodermal cells; 2) Identify interactions among components in the lignin polymerizing protein complex, and develop an initial structural model of the complex; and 3) establish that CS lignin in DP knockouts have molecular defects. We envisage a new paradigm for lignification, this being of critical importance to current BER/EMSL emphases, and that would enable informed development of bioenergy crops producing labile lignocellulosic biomass for the nascent biofuels/bioproducts industry.