Imaging Cellulose - Carbohydrate Binding Module Interactions With Nanometer Resolution Using Single Molecule Flourescence Methods

South Dakota School Of Mines And Technology, Rapid City SD

Investigators

Abstract

ID: MPS/DMR/BMAT(7623) 1206908 PI: Smith, Steve ORG: South Dakota School of Mines and Technology Title: Imaging Cellulose - Carbohydrate Binding Module Interactions with Nanometer Resolution Using Single Molecule Fluorescence Methods INTELLECTUAL MERIT: Cellulose, one of the most abundant biopolymers on earth, is a naturally occurring nanomaterial. It is the primary component of plant cell walls and the feedstock for valuable products derived from biomass sugars, such as liquid and gaseous fuels. Most of what is known about the chemical structure of biomass comes from analysis of the products generated during its decomposition via batch methods. These approaches necessarily lose the spatial correlations between the biomolecules that drive biomass degradation and the chemical-spatial structure of the feedstock, that is, the plant cell wall and its primary constituents, cellulose, hemi-cellulose, and lignin. This project will focus on exploiting single molecule, super-resolution imaging methods to reveal important facets of cellulose-carbohydrate binding module (CBM) interactions that are lost in the ensemble averaging of batch analyses. CBMs play a critical role in the action of a large family of cellulase enzymes, locating and positioning the catalytic components on their target substrates. This project will utilize single molecule fluorescence methods to reveal molecular level details of the interaction of families of CBMs, derived from microbial, bacterial, and cellulosomal sources, with cellulose substrates. The binding characteristics of single CBMs, including specificity, orientation, motility, and binding persistence will be studied using CBMs tagged with fluorescent proteins (FPs), including photo-activated FPs, to extract orientation and positional information at the single molecule level. Sequential imaging of sparse subsets of single molecules with high resolution imaging approaching the 5nm level will be used to study these processes and the spatio-chemical structure of biomass. BROADER IMPACTS: This project aims to provide detailed knowledge of cellulose-carbohydrate binding module interactions, thus building a biophysical knowledge of the molecular processes responsible for the breakdown of cellulose to simple sugars, knowledge critical to enabling new biotechnologies, which could accelerate conventional bioprocessing. Such knowledge could, for instance, aid in selecting new enzymes or designing enzymatic bioprocessing systems. This work thus leverages the significant efforts made by the State of South Dakota towards the utilization of biomass in the production of biofuels and other biomass-derived products, and investments made toward stimulating this industry. The proposed work will enrich graduate and undergraduate education in South Dakota by enabling collaborative research opportunities and interdisciplinary training for scientists and engineers in the region. SDSM&T also maintains a permanent Native American outreach program, with established and well-organized outreach programs such as SD GEAR UP, aimed at increasing the number of Native American high-school students continuing on to college. On-going aspects of this project will be incorporated into the educational portions of this program.