, Volume 16, Issue 4, pp 587–597 | Cite as

Does the cellulose-binding module move on the cellulose surface?

  • Yu-San Liu
  • Yining Zeng
  • Yonghua Luo
  • Qi Xu
  • Michael E. Himmel
  • Steve J. Smith
  • Shi-You Ding


Exoglucanases are key enzymes required for the efficient hydrolysis of crystalline cellulose. It has been proposed that exoglucanases hydrolyze cellulose chains in a processive manner to produce primarily cellobiose. Usually, two functional modules are involved in the processive mechanism: a catalytic module and a carbohydrate-binding module (CBM). In this report, single molecule tracking techniques were used to analyze the molecular motion of CBMs labeled with quantum dots (QDs) and bound to cellulose crystals. By tracking the single QD, we observed that the family 2 CBM from Acidothermus cellulolyticus (AcCBM2) exhibited linear motion along the long axis of the cellulose fiber. This apparent movement was observed consistently when different concentrations (25 μM to 25 nM) of AcCBM2 were used. Although the mechanism of AcCBM2 motion remains unknown, single-molecule spectroscopy has been demonstrated to be a promising tool for acquiring new fundamental understanding of cellulase action.


Cellulose Carbohydrate-binding module (CBM) Single molecule spectroscopy 



The authors thank Dr. Haw Yang and his group at University of California at Berkeley for valuable discussions. The authors gratefully acknowledge the US 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.


  1. Ai X, Xu Q, Jones M, Song Q, Ding SY, Ellingson RJ, Himmel M, Rumbles G (2007) Photophysics of (CdSe) ZnS colloidal quantum dots in an aqueous environment stabilized with amino acids and genetically-modified proteins. Photochem Photobiol Sci 6(9):1027–1033. doi: 10.1039/b706471c CrossRefGoogle Scholar
  2. Blainey PC, van Oijent AM, Banerjee A, Verdine GL, Xie XS (2006) A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA. Proc Natl Acad Sci USA 103(15):5752–5757. doi: 10.1073/pnas.0509723103 CrossRefGoogle Scholar
  3. Braslavsky I, Hebert B, Kartalov E, Quake SR (2003) Sequence information can be obtained from single DNA molecules. Proc Natl Acad Sci USA 100(7):3960–3964. doi: 10.1073/pnas.0230489100 CrossRefGoogle Scholar
  4. Cornish PV, Ha T (2007) A survey of single-molecule techniques in chemical biology. ACS Chem Biol 2(1):53–61. doi: 10.1021/cb600342a CrossRefGoogle Scholar
  5. Ding SY, Smith S, Xu Q, Sugiyama J, Jones M, Rumbles G, Bayer EA, Himmel ME (2005) Ordered arrays of quantum dots using cellulosomal proteins. Ind Biotechnol 1:198–206. doi: 10.1089/ind.2005.1.198 CrossRefGoogle Scholar
  6. Ding SY, Xu Q, Ali MK, Baker JO, Bayer EA, Barak Y, Lamed R, Sugiyama J, Rumbles G, Himmel ME (2006) Versatile derivatives of carbohydrate binding modules for imaging of complex carbohydrates approaching the molecular level of resolution. Biotechniques 41:435–443. doi: 10.2144/000112244 CrossRefGoogle Scholar
  7. Ding SY, Xu Q, Crowley M, Zeng Y, Nimlos M, Lamed R, Bayer EA, Himmel ME (2008) A biophysical perspective on the cellulosome: new opportunities for biomass conversion. Curr Opin Biotechnol 19(3):218–227. doi: 10.1016/j.copbio.2008.04.008 CrossRefGoogle Scholar
  8. Goldman ER, Medintz IL, Hayhurst A, Anderson GP, Mauro JM, Iverson BL, Georgiou G, Mattoussi H (2005) Self-assembled luminescent CdSe-ZnS quantum dot bioconjugates prepared using engineered poly-histidine terminated proteins. Anal Chim Acta 534(1):63–67. doi: 10.1016/j.aca.2004.03.079 CrossRefGoogle Scholar
  9. Gopich IV (2008) Concentration effects in “Single-Molecule” spectroscopy. J Phys Chem B 112(19):6214–6220. doi: 10.1021/jp0764182 CrossRefGoogle Scholar
  10. Ha T, Rasnik I, Cheng W, Babcock HP, Gauss GH, Lohman TM, Chu S (2002) Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419(6907):638–641. doi: 10.1038/nature01083 CrossRefGoogle Scholar
  11. Imai T, Putaux JL, Sugiyama J (2003) Geometric phase analysis of lattice images from algal cellulose microfibrils. Polymer 44(6):1871–1879. doi: 10.1016/S0032-3861(02)00861-3 CrossRefGoogle Scholar
  12. Jervis EJ, Haynes CA, Kilburn DG (1997) Surface diffusion of cellulases and their isolated binding domains on cellulose. J Biol Chem 272(38):24016–24023. doi: 10.1074/jbc.272.38.24016 CrossRefGoogle Scholar
  13. Kai Z, Hauyee C, Aihua F, Alivisatos AP, Haw Y (2006) Continuous distribution of emission states from single CdSe/ZnS quantum dots. Nano Lett 6(4):843–847. doi: 10.1021/nl060483q CrossRefGoogle Scholar
  14. Lakadamyali M, Rust MJ, Babcock HP, Zhuang XW (2003) Visualizing infection of individual influenza viruses. Proc Natl Acad Sci USA 100(16):9280–9285. doi: 10.1073/pnas.0832269100 CrossRefGoogle Scholar
  15. Levy I, Shoseyov O (2002) Cellulose-binding domains biotechnological applications. Biotechnol Adv 20(3–4):191–213. doi: 10.1016/S0734-9750(02)00006-X CrossRefGoogle Scholar
  16. Moerner WE (2007) Single-molecule chemistry and biology special feature: new directions in single-molecule imaging and analysis. Proc Natl Acad Sci USA 104(31):12596–12602. doi: 10.1073/pnas.0610081104 CrossRefGoogle Scholar
  17. Morag E, Lapidot A, Govorko D, Lamed R, Wilchek M, Bayer EA, Shoham Y (1995) Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Appl Environ Microbiol 61(5):1980–1986Google Scholar
  18. Nan XL, Sims PA, Chen P, Xie XS (2005) Observation of individual microtubule motor steps in living cells with endocytosed quantum dots. J Phys Chem B 109(51):24220–24224. doi: 10.1021/jp056360w CrossRefGoogle Scholar
  19. Okten Z, Churchman LS, Rock RS, Spudich JA (2004) Myosin VI walks hand-over-hand along actin. Nat Struct Mol Biol 11(9):884–887. doi: 10.1038/nsmb815 CrossRefGoogle Scholar
  20. Schuler B, Lipman EA, Eaton WA (2002) Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Nature 419(6908):743–747. doi: 10.1038/nature01060 CrossRefGoogle Scholar
  21. Silver J, Ou W (2005) Photoactivation of quantum dot fluorescence following endocytosis. Nano Lett 5(7):1445–1449. doi: 10.1021/nl050808n CrossRefGoogle Scholar
  22. Slocik JM, Moore JT, Wright DW (2002) Monoclonal antibody recognition of histidine-rich peptide encapsulated nanoclusters. Nano Lett 2(3):169–173. doi: 10.1021/nl015706l CrossRefGoogle Scholar
  23. Steinmeyer R, Noskov A, Krasel C, Weber I, Dees C, Harms GS (2005) Improved fluorescent proteins for single-molecule research in molecular tracking and co-localization. J Fluoresc 15(5):707–721. doi: 10.1007/s10895-005-2978-4 CrossRefGoogle Scholar
  24. Sun YH, Liu YS, Vernier PT, Liang CH, Chong SY, Marcu L, Gundersen MA (2006) Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells. Nanotechnology 17(17):4469–4476. doi: 10.1088/0957-4484/17/17/031 CrossRefGoogle Scholar
  25. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783. doi: 10.1016/S0006-3495(02)75618-X CrossRefGoogle Scholar
  26. Tormo J, Lamed R, Chirino AJ, Morag E, Bayer EA, Shoham Y, Steitz TA (1996) Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. EMBO J 15(21):5739–5751Google Scholar
  27. Vrljic M, Nishimura SY, Moerner WE, McConnell HM (2005) Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane. Biophys J 88(1):334–347. doi: 10.1529/biophysj.104.045989 CrossRefGoogle Scholar
  28. Warshaw DM, Kennedy GG, Work SS, Krementsova EB, Beck S, Trybus KM (2005) Differential Labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity. Biophys J 88(5):L30–L32. doi: 10.1529/biophysj.105.061903 CrossRefGoogle Scholar
  29. Xu Q, Tucker MP, Arenkiel P, Ai X, Rumbles G, Sugiyama J, Himmel ME, Ding SY (2009) Labeling the planar face of crystalline cellulose using quantum dots directed by type-I carbohydrate-binding modules. Cellulose 16(1):19–26Google Scholar
  30. Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065. doi: 10.1126/science.1084398 CrossRefGoogle Scholar
  31. Yildiz A, Park H, Safer D, Yang ZH, Chen LQ, Selvin PR, Sweeney HL (2004a) Myosin VI steps via a hand-over-hand mechanism with its lever arm undergoing fluctuations when attached to actin. J Biol Chem 279(36):37223–37226. doi: 10.1074/jbc.C400252200 CrossRefGoogle Scholar
  32. Yildiz A, Tomishige M, Vale RD, Selvin PR (2004b) Kinesin walks hand-over-hand. Science 303(5658):676–678. doi: 10.1126/science.1093753 CrossRefGoogle Scholar
  33. Yu WW, Qu LH, Guo WZ, Peng XG (2003) Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals. Chem Mater 15(14):2854–2860. doi: 10.1021/cm034081k CrossRefGoogle Scholar
  34. Zhuang XW, Rief M (2003) Single-molecule folding. Curr Opin Struct Biol 13(1):88–97. doi: 10.1016/S0959-440X(03)00011-3 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Yu-San Liu
    • 1
  • Yining Zeng
    • 1
  • Yonghua Luo
    • 1
  • Qi Xu
    • 1
  • Michael E. Himmel
    • 1
  • Steve J. Smith
    • 2
  • Shi-You Ding
    • 1
  1. 1.Chemical and Biosciences CenterNational Renewable Energy LaboratoryGoldenUSA
  2. 2.Department of Electrical Engineering and PhysicsSouth Dakota School of MinesRapid CityUSA

Personalised recommendations