Development of an Integrated Structural Mass Spectrometry Platform for Unveiling the Structure of an Unknown Protein Complex Machinery in Lignin Synthesis
EMSL Project ID
50427
Abstract
Multiple distinct Lignin-Forming Complexes (LFC) are proposed to be templates and catalysts that orchestrate lignin synthesis in plants. It is hypothesized that each LFC consists of one or more oxidases, transmembrane templating proteins, and monolignol radical-binding "dirigent" proteins. Yet no direct experimental evidence is available so far to support the putative structure. A LFC from the genus Forsythia was the first and only such system to be isolated as an intact complex. We herein propose to determine the composition and the architecture of this LFC at the molecular level to understand aspects of plant growth and development relevant to engineering of innovative solutions for improving lignocellulosic biomass utilization and environmental carbon transformation. The endogenous LFC is heterogeneous with unknown components and is challenging to study by classical structural biology methods based on previous analysis. We aim to first use advanced top-down mass spectrometry capability at EMSL to identify the unknown components of LFC through de novo sequencing because the complete sequenced genome for Forsythia is not available. We will then utilize native mass spectrometry to rapidly and effectively measure stoichiometry of subunits. In addition, we will develop chemical labeling methods to probe protein surface residues and interaction interfaces. All mass spectrometry data will be integrated with computational methods to generate models of LFC. To obtain high-resolution structure of LFC, we will use recently established cell-free expression system at EMSL to generate recombinant LFC, characterize them by native mass spectrometry, and select suitable targets for further analysis by crystallography and cryo-electron microscopy.The structural work will provide definitive experimental evidence to support the theory that lignin synthesis can be mediated by proteins. It will open up unprecedented possibilities of engineering plants by modifying the biologically controlled lignification pathways, including production of biomass that is a more efficient source of biofuels. The method development work in this study will also integrate multiple emerging capabilities, including native mass spectrometry, cell-free expression, and cryo-electron microscopy for structural biology applications. The proposed mass spectrometry workflow is expected to complement existing and emerging capabilities at EMSL for analysis of unknown proteins. Endogenous protein complexes may be dynamic, heterogeneous, post-translationally modified (e.g. glycosylated), or partially unfolded. New capabilities enabling rapid structural analysis of these complex systems would be indispensable to increase the throughput and help establish a foundation for EMSL to make important contributions in structural biology relevant to bioenergy.
Project Details
Start Date
2018-10-11
End Date
2020-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members