and Dong Yang
Fluorescence spectroscopy techniques allow the visualization of molecular events that are not readily accessible through other methods. Fluorescence emission enhances the detection and spatial and temporal resolution of binding and molecular displacements. In this area, we are exploiting the advantages offered by fluorescence spectroscopy to study the interactions between cellulases and cellulose fibrils at the most fundamental scales spanning the micro to nanometer range. To this end we have developed methods to immobilize cellulose on glass surfaces that allow the controlled observation of these molecular events (1). We have also developed fluorescence labeling and purification protocols that have enabled us to recover labeled enzymes with well defined catalytic and spectroscopic characteristics (2). Described below are some examples of the studies performed using our fluorescence cellulase-cellulose system.
Bacterial microcrystalline cellulose (BMCC) is immobilized on solid surfaces through a polymer lift-off technique. A glass surface was coated with a polymer that was then photolithographically patterned to expose select areas of the underlying glass surface. BMCC was then applied to the surface. After the polymer is removed by peel-off, cellulose only remains in the exposed areas. Cellulose features are closely related to the size of the patterns used to immobilize the cellulose.
Immobilized cellulose fibrils have been used to study the binding kinetics of fluorescently-labeled cellulases with high spatial and temporal resolution (1). In this study, the use of molecules labeled with different fluorophores has allowed us to determine the binding behavior of cellulases to cellulose fibrils, mats and particles. We have observed that the binding proceeds according to a binding saturation model in cellulose fibrils, but deviates substantially for more complex structures. These results point towards the important role of physical structure and pore distribution in binding of cellulases. With this system we are also investigating the binding behavior of cellulases in cocktails.
Images of AF647 labeled cellulases (red, center) binding on DTAF-labeled cellulose (green, left) can help determine the accessibility of cellulose structures to the enzymes. An overlay of the fluorescence (right images) of both elements shows areas where cellulases bind readily (in yellow and red) and those that are not easily accessible (in green).
The isolation of highly labeled fluorescent cellulases with no loss in activity has enabled us to probe the cellulase-cellulose interactions at the single molecule level. Using Total Internal Reflection Fluorescence Microscopy coupled with a highly sensitive EM-CCD camera, pictures can be taken where single cellulases can be identified bound to cellulose fibrils. These images, taken in rapid succession give us a time course description of the cellulase displacement on the cellulose fibrils. The high numbers of photons emitted by the labeled cellulases also allow localization of the center of mass of each cellulase with precision that surpasses that of the diffraction limit of the optical microscope. In this way, cellulases can be localized with a precision close to their molecular size (10-30nm), and their processivity estimated from molecular displacements.
Single Cel9A cellulases (red) bound on cellulose microfibrils can be imaged with high spatial and temporal resolution using a EM-CCD camera coupled to a TIRF microscope.
A key to improving the efficiency of biomass conversion for the production of biofuels is understanding the interactions between cell wall degrading enzymes and real substrates (biomass particles). Pretreatment of substrates to achieve the release of hemicellulose and lignin changes the accessibility of the crystalline cellulose to the enzymes. To assess the changes in accessibility in cellulose particles we are utilizing fluorescently labeled cellulases and confocal microscopy to look at the three dimensional spatial distribution of cellulases bound to cellulose particles. With confocal microscopy we can image of thin slices of the cellulose particles, and through axial scanning reconstruct the spatial distribution of fluorescence. These experiments will enable the distinction between easily accessible surfaces for cellulase binding and restricted volumes where enzymes cannot penetrate.
Z-projection of the three dimensional reconstruction of fluorescence distribution in a pretreated cellulose particle. Autofluorescence is used to image the particle (blue) and identify the sites where cellulases bind more readily (green).
(1) Moran-Mirabal, J. M., Santhanam, N., Corgie, S. C., Craighead, H. G., and Walker, L. P. 2008. Immobilization of Cellulose Fibrils on Solid Substrates for Cellulase Binding Studies Through Quantitative Fluorescence Microscopy. Biotechnology Bioengineering, 101(6): 1129-1141.
(2) Moran-Mirabal, J. M., Corgie, S. C. , Bolewski, J. C., Smith, H. M., Cipriany, B. R., Craighead, H. G.,Walker, L. P. 2009. Labeling and purification of cellulose-binding proteins for high resolution fluorescence applications. Analytical Chemistry, 81: 7981-7987.