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Single Molecule Studies of Cellulose-Binding Enzymes

Benjamin R. Cipriany, Jose M. Moran-Mirabal*, Harold G. Craighead, and Larry Walker*

*Department of Biological and Environmental Engineering, Cornell

Abstract

Figure 1: Microfluidic channel with a constricted, submicrometer cross-section region. Labeled cellulases flow through the channel and across a laser beam that impinges the substrate perpendicular to the channel, causing fluorescence to occur. The scale bar is 10 micrometers.

Enzymatic depolymerization of cellulose is an essential process in the production of biofuels. Single molecule fluorescence studies of this process would facilitate increased understanding of cellulose saccharification and lead to more efficient design of enzymatic cocktails for biofuel production. Fluorescence labeling techniques can hinder the catalytic function of the cellulase protein by modifying active sites. We demonstrate a method for fluorescent-label attachment to cellulase, which retains wild-type functionality of the protein and yields fluorescently bright, high-purity labeling. The spectroscopic characteristics of fluorescently-labeled enzymes are studied using fused-silica sub-micrometer fluidic channels to isolate cellulase molecules during laser induced fluorescence. We show that unbound fluorescent dye is successfully removed using solid-phase purification, providing a highly homogeneous population of cellulases with up to six dyes per cellulase. It is also concluded that the addition of more than six fluorophores per cellulase hinders the total photon output per cellulase. This method can be adapted to label any mixture of labeled proteins and we believe it will lead to improved visualization of cellulase-cellulose interactions at the single molecule level.

Research Summary

Submicrometer fluidic channels (cross-section 250nm x 500nm) were constructed in a fused silica substrate using - a single layer of photolithography followed by reactive ion etching [Figure 1]. Fluid reservoirs access the channels by through-wafer ports and the final device is assembled with a direct wafer bond technique. The process allows for rapid prototyping of many fluid channel arrays on a 100mm substrate. Cellulase proteins labeled with Alexa Fluor 647 were labeled and purified through solid phase methods detailed in [1]. Proteins were suspended in a phosphate buffered saline solution and flowed through the fluidic channels using electrokinetic drive. The fluorescence from each purified population of labeled cellulases was measured using a collection of approximately 10,000 individual molecules. We studied impact of the degree of labeling (DOL) on photon output per cellulase for labeled populations with a number of attached fluorophores between 1 and 6 [Figure 2]. The observed flow velocity of the proteins increased proportional to the number of fluorescent dye attached (data not shown), due to the increased effective charge to mass ratio. The average number of photons collected from each cellulase was found to increase linearly for DOL <= 4 and then exhibited quenching effects at DOL > 4 [Figure 3]. These catalytically-active, labeled cellulase molecules can be used in single molecule studies of cellulase-cellulose interactions with the confidence that they reflect the wild-type catalytic behavior and have high photon output.

Figure 2: Photon counting histogram illustrates the total number of photons collected from each single molecule observation. The mean fluorescence increases proportional to the degree of labeling.

Figure 3: Samples with a higher degree of labeling produce more fluorescence per cellulase molecule, until the onset of fluorescence quenching effects for DOL > 4.

References

  1. "Labeling and Purification of Cellulose Binding Enzymes Retaining Wild-Type Functionality for High-Resolution Fluorescence Applications." Jose M. Moran-Mirabal, Stephane C. Corgie, Jacob C. Bolewski, Hanna M. Smith, Benjamin R. Cipriany, Harold G. Craighead, and Larry P. Walker. Proceedings of the National Academy of Sciences, In Review.
  2. Mathieu Foquet, Jonas Korlach, Warren R. Zipfel, Watt W. Webb, Harold G. Craighead, "Focal Volume Confinement by Submicrometer-Sized Fluidic Channels,”" Analytical Chemistry, vol. 76, pg. 1618-1626, 2004. Abstract
  3. S. Stavis, J. Edel, K. Samiee, and H. Craighead. "Single Molecules Studies of Quantum Dot Conjugates in a Submicrometer Fluidic Channel," Lab on a Chip vol. 5, 337-343 (2005). Abstract