DOPI and PALM imaging of single carbohydrate binding modules bound to cellulose nanocrystals

We use single molecule imaging methods to study the binding characteristics of carbohydrate-binding modules (CBMs) to cellulose crystals. The CBMs are carbohydrate specific binding proteins, and a functional component of most cellulase enzymes, which in turn hydrolyze cellulose, releasing simple sugars suitable for fermentation to biofuels. The CBM plays the important role of locating the crystalline face of cellulose, a critical step in cellulase action. A biophysical understanding of the CBM action aids in developing a mechanistic picture of the cellulase enzyme, important for selection and potential modification. Towards this end, we have genetically modified cellulose-binding CBM derived from bacterial source with green fluorescent protein (GFP), and photo-activated fluorescence protein PAmCherry tags, respectively. Using the single molecule method known as Defocused Orientation and Position Imaging (DOPI), we observe a preferred orientation of the CBM-GFP complex relative to the Valonia cellulose nanocrystals. Subsequent analysis showed the CBMs bind to the opposite hydrophobic faces of the cellulose nanocrystals with a welldefined cross-orientation of about ~ 70°. Photo Activated Localization Microscopy (PALM) is used to localize CBMPAmCherry with a localization accuracy of ~ 10nm. Analysis of the nearest neighbor distributions along and perpendicular to the cellulose nanocrystal axes are consistent with single-file CBM binding along the fiber axis, and microfibril bundles consisting of close packed ~ 20nm or smaller cellulose microfibrils.