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

ID：MPS/DMR/BMAT(7623)1206908 PI：Smith，Steve ORG：南达科他州矿业与技术学院标题：使用单分子荧光方法成像具有纳米分辨率的纤维素-碳水化合物结合模块相互作用INTELLECTUCTUAL优点：纤维素是地球上最丰富的生物聚合物之一，是一种天然存在的纳米材料。它是植物细胞壁的主要成分，也是从生物质糖中提取有价值产品的原料，如液体和气态燃料。人们对生物质化学结构的大部分了解都是通过批量方法对其分解过程中产生的产物进行分析。这些方法必然会失去驱动生物质降解的生物分子与原料的化学空间结构之间的空间相关性，即植物细胞壁及其主要成分纤维素、半纤维素和木质素。该项目将专注于开发单分子、超分辨率成像方法，以揭示在批量分析的总体平均中丢失的纤维素-碳水化合物结合模块(CBM)相互作用的重要方面。CBMS在一大类纤维素酶的作用中起着关键作用，它定位和定位目标底物上的催化成分。该项目将利用单分子荧光方法揭示来自微生物、细菌和纤维素体来源的CBM家族与纤维素底物相互作用的分子水平细节。单个CBM的结合特性，包括特异性、方向性、运动性和结合持久性，将使用标记有荧光蛋白(FP)的CBM进行研究，以在单分子水平上提取方向和位置信息。单分子稀疏子集的顺序成像将被用于研究这些过程和生物体的空间化学结构，高分辨率成像接近5 nm水平。BROADER影响：该项目旨在提供纤维素-碳水化合物结合模块相互作用的详细知识，从而建立负责将纤维素分解为单糖的分子过程的生物物理知识，这些知识对于实现新的生物技术至关重要，可以加速传统的生物加工。例如，这些知识可以帮助选择新的酶或设计酶生物处理系统。因此，这项工作充分利用了南达科他州在生产生物燃料和其他生物质衍生产品中利用生物质所作的重大努力，以及为刺激这一行业而进行的投资。拟议的工作将丰富南达科他州的研究生和本科教育，为该地区的科学家和工程师提供合作研究机会和跨学科培训。SDSM&amp；T还维持着一个永久性的美洲原住民外展计划，建立并组织良好的外展计划，如SD Gear Up，旨在增加继续上大学的美国原住民高中生的数量。这个项目正在进行的方面将被纳入这个项目的教育部分。

ID: MPS/DMR/BMAT(7623) 1206908 PI: Smith, Steve ORG: South Dakota School of Mines and TechnologyTitle: Imaging Cellulose - Carbohydrate Binding Module Interactions with Nanometer Resolution Using Single Molecule Fluorescence MethodsINTELLECTUAL 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.