Understanding Enzyme Activity Using Single Molecule TrackingYu-San (Angela) Liu, Yining Zeng, Yonghua Luo, Qi Xu, Mike Himmel, Steve Smith*, Hui Wei, and Shi-You Ding

Chemical & Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 *South Dakota School of Mines and Technology, Rapid City, SD 57701

Approach1:Tracking Quantum Dots (QDs)-labeled CBM Analysis: Image processing sequence

White light image of  Valonia fibers

Model of cellulose/CBM/QD system

Total Internal Reflection Fluorescence Microscopy (TIRF‐M) is used to measure focused and defocused fluorescenceof fluorescent‐tagged CBMs. The goal of this study isunderstanding the dynamic behavior of enzymes.

Single Molecule Tracking and TIRFSingle Molecule Tracking and TIRFSingle Molecule Tracking and TIRFSingle Molecule Tracking and TIRF Does CBM Move on Cellulose?Does CBM Move on Cellulose?Does CBM Move on Cellulose?Does CBM Move on Cellulose? How does CBM Bind to Cellulose?How does CBM Bind to Cellulose?How does CBM Bind to Cellulose?How does CBM Bind to Cellulose?

TIRF system utilizesthis evanescent waveto excite singlemolecules in the thinsection (<100 nm),thus producefluorescence imageswith high signal‐to‐noise (S/N) ratio

Incident lightReflected light

High refractive index(coverglass)

Low refractive index(specimen)

Incident angles greater than critical angle

Evanescent wave,typically less than 100 nm

Defocused fluorescence image

Fluorescence image of GFP‐labeled CBMs bound to these Valonia fibers

Model of cellulose/CBM/QD system  

QD are anchored to the CBM by the

Four QDs lined up on the cellulose 

understanding the dynamic behavior of enzymes.

noise (S/N) ratio.500 nm

 Trajectory showed CBM movingalong the direction of the cellulose Although QDs are bright, theemission intermittency (blinking)is difficult to distinguish one QD

Time (s)Simulated defocused orientational and positional image (DOPI )

dual histidine‐tags (His‐tag) andfused at N‐and C‐termini

Schematic of TIRF system

Approach2:Tracking GFP-labeled CBM

is difficult to distinguish one QDmoving or two QDs blinking

GFP

 Preliminary results suggest that GFP‐CBM preferentially bind perpendicular to the fiber

3‐D Reconstruction

Model of GFP labeled CBM bound on cellulose  For the geometry of imaging system (top), with slight movement ofthe optics toward the molecule, the image can be defocused to yieldcharacteristic interference patterns (bottom). From the pattern, weinfer the molecule orientation.

CDLinker

Dipole orientation

CBM

Linker

GFP has chromophore in itscenter with a fluorescencedipole allowing us to study thesingle molecule orientation

Cellulase and CBMCellulase and CBMCellulase and CBMCellulase and CBM

Carbohydrate‐binding module (CBM):Bind to the substrate

Catalytic domain (CD):

Model of cellulase depolymerizing cellulose 

Cellulosome AssemblyCellulosome AssemblyCellulosome AssemblyCellulosome Assembly

E E Catalytic subunits fused with GFP or RFP

Model of cellulosome GFP RFP

Cellulose surface

Family 1 CBM  Family 2 CBM Family 3 CBM

2 μm

50 0

2

4

6

8

10

12

14Time (s)

white light

TIRFCBM binding on cellulose is specific   We have developed three families of enzyme (GH5, GH12, GH74)

Catalytic domain (CD):Hydrolyze/depolymerize carbohydrate polymers

Type A Surface binding CBMs — This class of CBM binds to insoluble, highly crystalline cellulose.  Three families were used in this study.

Cohesin

Dockerin

CBM Carbohydrate‐binding module

CBMwhite lightoverlay

The BioEnergy Science Center (BESC) is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science 

y(from T. reesei CBHI)36 amino acids, 

3 nm x 1.8 nm x 1 nm

y(from C. fimi)

110 amino acids, 4.5 nm x 2.5 nm x 2.5 nm

y( from C. thermocellum)

155 amino acids, 3 nm x 3 nm x 4.5 nm

The authors gratefully acknowledge the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy Biomass Program for support of the work to develop quantum dot conjugates and support from the DOE Office of Science, Office of Biological and Environmental Research through the BioEnergy Science Center (BESC), a DOE Bioenergy Research Center, for the work on single molecule visualization and analysis.Presented at the Bioenergy Sciences Center (BESC) Annual Retreat, 22-24 June 2009, Asheville, North Carolina

 CBM moving along the cellulose direction

p y ( )labeled with GFP or RFP. Future work includes applying singlemolecule imaging technique to study cellulosome structure aswell as dynamic behavior.

NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC. • NREL/PO-270-46339

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