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

ID：MPS/DMR/BMAT（7623）1206908 PI：史密斯，史蒂夫 ORG：南达科他州矿业与技术学院题目：使用单分子荧光方法以纳米分辨率成像纤维素-碳水化合物结合模块相互作用智力优势：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，也是从生物质糖衍生的有价值产品的原料，如液体和气体燃料。 大多数关于生物质化学结构的知识来自于通过分批方法对其分解过程中产生的产物的分析。 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（即植物细胞壁及其主要成分纤维素、半纤维素和木质素）之间的空间相关性。 该项目将专注于利用单分子，超分辨率成像方法来揭示纤维素-碳水化合物结合模块（CBM）相互作用的重要方面，这些方面在批量分析的整体平均中丢失。 CBM在纤维素酶大家族的作用中起关键作用，将催化组分定位和定位在其靶底物上。 该项目将利用单分子荧光方法来揭示来自微生物、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。 将使用标记有荧光蛋白（FP）（包括光活化FP）的CBM研究单个CBM的结合特性，包括特异性、方向性、运动性和结合持久性，以提取单分子水平的方向和位置信息。 单分子稀疏子集的连续成像与接近5 nm水平的高分辨率成像将用于研究这些过程和生物质的空间化学结构。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，从而建立负责纤维素分解为单糖的分子过程的生物物理知识，知识对实现新的生物技术至关重要，这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加工系统。 因此，这项工作充分利用了南达科他州为利用生物质生产生物燃料和其他生物质衍生产品所做的重大努力，以及为刺激这一行业所做的投资。 拟议的工作将丰富南达科他州的研究生和本科生教育，使该地区的科学家和工程师的合作研究机会和跨学科培训。 SDSM T还保持着一个永久的美洲原住民外展计划，建立和组织良好的外展计划，如SD GEAR UP，旨在增加美洲原住民高中学生继续上大学的人数。 该项目正在进行的方面将纳入该方案的教育部分。

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.