Gransker Receptor-ligandsysternene med Cellulosome med AFM-baserte Single-molekyl kraft spektroskopi
Summary
Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.
Abstract
Cellulosomes er diskrete Multienzyme komplekser som brukes av en undergruppe av anaerobe bakterier og sopp å fordøye lignocelluloseholdige substrater. Montering av enzymer bort på noncatalytic scaffoldprotein er regissert av samhandling mellom en familie av beslektede reseptor-ligand par som omfatter samspill cohesin og dockerin moduler. Den ekstremt sterk binding mellom cohesin og dockerin moduler resultater i dissosiasjonskonstanter i den lave picomolar til nanomolarområdet, noe som kan hemme nøyaktige off-rate målinger med konvensjonelle bulk metoder. Single-molekyl kraft spektroskopi (SMFs) med atommikroskop måler responsen til den enkelte biomolekyler for å tvinge, og i motsetning til andre enkelt molekyl manipuleringsmetoder (dvs. optisk pinsetter), er optimal for å studere høyaffinitets-reseptor-ligand-vekselvirkninger, fordi av sin evne til å sondere høy styrke regime (> 120 pN). Her presenterer vi vår fullstendige protokollen for å studere cellulosomal protein forsamlinger på enkelt-molekyl nivå. Ved hjelp av et protein topologi avledet fra de innfødte cellulosome, vi jobbet med enzym-dockerin og karbohydrat bindende modul cohesin (CBM-cohesin) fusjon proteiner, hver med en tilgjengelig gratis tiolgruppe på en konstruert cysteinrest. Vi presenterer sete-spesifikk overflate immobilisering protokoll, sammen med vår måling og analyse av data Fremgangsmåten for å oppnå detaljerte bindingsparametre for den høyaffinitets-kompleks. Vi viser hvordan du kvantifisere enkeltunderdomene utfolder krefter, komplekse bruddstyrker, kinetiske off-priser, og potensielle bredder på bindingen godt. Vellykket bruk av disse metodene i karakteriserer cohesin-dockerin interaksjon ansvarlig for montering av multidomain cellulolytic komplekser er nærmere beskrevet.
Introduction
Cellulosomes are large multienzyme complexes displayed on the surface of anaerobic cellulolytic bacteria (e.g. C. thermocellum) that have evolved to efficiently depolymerize plant cell wall lignocellulose into soluble oligosaccharides1. A central attribute of cellulosomes is the high-affinity cohesin-dockerin interaction. In the most prominent paradigm, a highly conserved 60-75 amino acid type I dockerin module is displayed at the C-terminal end of the various bacterial enzymes. The dockerin module directs assembly of synergistic combinations of enzymes onto the noncatalytic scaffold protein ('scaffoldin'), which comprises a polyprotein of cohesin domains that are specific for the type I dockerin module. At higher levels, cellulosome architecture can become very complex, incorporating alternative cohesin and dockerin pairs (e.g. type II, type III) that anchor the structures to the cell surface and allow for the assembly of branched structures containing multiple scaffoldins2. The various cohesin-dockerin types, despite having related structures, exhibit differential binding specificities suppressing cross reactivity with unintended scaffoldins or components from other cellulosome-producing bacterial species. While bioinformatic approaches have successfully identified thousands of unique cellulosomal components at the genetic level, comparatively few protein structures are known, and the mechanisms at work in cohesin-dockerin specificity determination remains an active area of structural biology research.
Since the invention of the atomic force microscope (AFM) by Binnig et al.3, various AFM operational modes have been developed and continuously improved, including noncontact imaging, oscillation mode imaging4, and single molecule force spectroscopy (SMFS)5,6. SMFS has evolved into a widely used technique to directly probe individual proteins7-11, nucleic acids12-15, and synthetic polymers16-19. In a typical SMFS experiment to investigate receptor-ligand binding20,21, an AFM cantilever tip is modified with one of the binding partners, while a flat glass surface is modified with the complementary binding partner. The modified cantilever is brought into contact with the surface allowing the partners to bind. The base of the cantilever is then withdrawn at constant speed and the force is measured using the optical lever deflection method. The resultant force-distance data traces exhibit sawtooth-like peaks if binding was established. In cases where the binding partners are fused to multiple protein domains, each peak in the force-distance trace can be correlated to the unfolding of a single protein domain or folded subdomain, while the last peak corresponds to rupture of the protein binding interface. The specific positions of the force-resistant elements can be used as a fingerprint to identify the various protein domains of interest. This method can be used to interrogate important amino acids involved in protein folding and stabilization. Many models have been reported in the literature to treat the characteristic force extension behavior observed in SMFS experiments. The most commonly used models include the freely jointed chain (FJC) model22, the worm-like chain (WLC) model18,23-25, and the freely rotating chain (FRC) model25,26.
In our prior work11, we used single-molecule force spectroscopy to investigate the interaction of cohesin and dockerin modules. Here, we present an experimental protocol for glass surface and cantilever functionalization with enzyme-dockerin and CBM-cohesin protein constructs. We also present an AFM-based SMFS protocol including data acquisition and analysis procedures. The described protocol can easily be generalized to other molecular systems, and should prove particularly useful to researchers interested in high-affinity receptor ligand pairs.
Protocol
Representative Results
Discussion
To obtain meaningful data from single molecule force spectroscopy experiments, it is crucial to achieve well-defined and reproducible pulling geometries. The protocol used here results in site-specific immobilization of protein complexes in a defined pulling geometry.
The cantilevers used in this study were chosen due to their force sensitivity and high resonance frequency in water. Moreover, the small tip curvature of approximately 10 nm is advantageous for single molecule experiments due to …
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge funding from a European Research Council advanced grant to Hermann Gaub. Michael A. Nash gratefully acknowledges funding from Society in Science – The Branco Weiss Fellowship program. The authors thank Edward A. Bayer, Yoav Barak, and Daniel B. Fried at the Weizmann Institute of Science for generously providing the proteins used in this study. The authors thank Hermann E. Gaub, Elias M. Puchner, and Stefan W. Stahl for helpful discussions.
Materials
| 3-Aminopropyl dimethyl ethoxysilane | ABCR GmbH | AB110423 | |
| 5 kDa NHS-PEG-maleimide | Rapp Polymer | 13 5000-65-35 | |
| TCEP Disulfide reducing gel | Thermo Scientific, Pierce | 77712 | www.thermoscientific.com/pierce |
| Tris(hydroxymethyl)aminomethane | |||
| BioLever mini silicon nitride cantilevers | Olympus | BL-AC40TS-C2 | Soft batches |
| XYZ Piezoelectric actuators | Physik Instrumente GmbH | ||
| Infrared “broad spectrum” IR laser | Superlum | ||
| MFP-3D AFM Controller | Asylum Research | ||
| Igor Pro 6.31 | Wavemetrics | Data acquisition and analysis | |
| Sodium chloride | |||
| Calcium chloride | |||
| pH Meter | |||
| Sodium borate | |||
| Tweezers | |||
| Cover glasses | Thermo Scientific, Menzel-Gläser | 24 mm diameter, 0.5 mm thickness | |
| PTFE sample holder | custom made | ||
| Sonicator bath | |||
| Ethanol | analytical purity | ||
| Sulfuric acid (concentrated) | analytical purity | ||
| Hydrogen peroxide (30%) | analytical purity | ||
| Orbital shaker | |||
| Toluene | analytical purity | ||
| Filter paper | |||
| Glass slides | |||
| Microtubes | |||
| Micropipettes | |||
| Centrifuge | suitable for microtubes | ||
| Rotator | |||
| Petri dishes | |||
| Beakers | |||
| Optical microscope |
Referências
- Bayer, E. A., Belaich, J. P., Shoham, Y., Lamed, R. The cellulosomes: Multienzyme machines for degradation of plant cell wall polysaccharides. Annu. Rev. Microbiol. 58, 521-554 (2004).
- Bayer, E. A., Lamed, R., White, B. A., Flint, H. J. From Cellulosomes to Cellulosomics. Chem. Rec. 8 (6), 364-377 (2008).
- Binnig, G., Quate, C. F. Atomic Force Microscope. Phys. Rev. Lett. 56 (9), 930-933 (1986).
- Garcı́a, R., Perez, R. Dynamic atomic force microscopy methods. Surf. Sci. Rep. 47 (6), 197-301 (2002).
- Engel, A., Müller, D. J. Observing single biomolecules at work with the atomic force microscope. Nat. Struct. Biol. 7 (9), 715-718 (2000).
- Li, H., Cao, Y. Protein Mechanics: From Single Molecules to Functional Biomaterials. Acc. Chem. Res. 43 (10), 1331-1341 (2010).
- Florin, E. -. L., Moy, V. T., Gaub, H. E. Adhesion forces between individual ligand-receptor pairs. Science. 264 (5157), 415-417 (1994).
- Oberhauser, A., Hansma, P., Carrion-Vazquez, M., Fernandez, J. Stepwise unfolding of titin under force-clamp atomic force microscopy. Proc. Natl. Acad. Sci. U.S.A. 98 (2), 468-472 (2001).
- Puchner, E. M., Gaub, H. E. Force and function: probing proteins with AFM-based force spectroscopy. Curr. Opin. Struct. Biol. 19 (5), 605-614 (2009).
- Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J., Gaub, H. Reversible unfolding of individual titin immunoglobulin domains by AFM. Sci. 276 (5315), 1109-1112 (1997).
- Stahl, S. W., Nash, M. A., et al. Single-molecule dissection of the high-affinity cohesin–dockerin complex. Proc. Natl. Acad. Sci. U.S.A. 109 (50), 20431-20436 (2012).
- Boland, T., Ratner, B. D. Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy. Proc. Natl. Acad. Sci. U.S.A. 92 (12), 5297-5301 (1995).
- Morfill, J., Kühner, F., Blank, K., Lugmaier, R. A., Sedlmair, J., Gaub, H. E. B-S Transition in Short Oligonucleotides. Biophys. J. 93 (7), 2400-2409 (2007).
- Rief, M., Clausen-Schaumann, H., Gaub, H. E. Sequence-dependent mechanics of single DNA molecules. Nat. Struct. Biol. 6 (4), 346-350 (1999).
- Severin, P. M. D., Zou, X., Gaub, H. E., Schulten, K. Cytosine methylation alters DNA mechanical properties. Nucleic Acids Res. 39 (20), 8740-8751 (2011).
- Geisler, M., Balzer, B. N., Hugel, T. Polymer Adhesion at the Solid-Liquid Interface Probed by a Single-Molecule Force Sensor. Small. 5 (24), 2864-2869 (2009).
- Giannotti, M. I., Vancso, G. J. Interrogation of Single Synthetic Polymer Chains and Polysaccharides by AFM-Based Force Spectroscopy. Chem. Phys. Chem. 8 (16), 2290-2307 (2007).
- Hugel, T., Rief, M., Seitz, M., Gaub, H., Netz, R. Highly Stretched Single Polymers: Atomic-Force-Microscope Experiments Versus Ab-Initio Theory. Phys. Rev. Lett. 94 (4), 10-1103 (2005).
- Nash, M. A., Gaub, H. E. Single-Molecule Adhesion of a Stimuli-Responsive Oligo(ethylene glycol) Copolymer to Gold. ACS Nano. 6 (12), 10735-10742 (2012).
- Merkel, R., Nassoy, P., Leung, A., Ritchie, K., Evans, E. Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature. 397 (6714), 50-53 (1999).
- Morfill, J., Neumann, J., et al. Force-based Analysis of Multidimensional Energy Landscapes: Application of Dynamic Force Spectroscopy and Steered Molecular Dynamics Simulations to an Antibody Fragment–Peptide. Complex. J. Mol. Biol. 381 (5), 1253-1266 (2008).
- Ortiz, C., Hadziioannou, G. Entropic Elasticity of Single Polymer Chains of Poly(methacrylic acid) Measured by Atomic Force Microscopy. Macromolecules. 32 (3), 780-787 (1999).
- Bustamante, C., Marko, J. F., Siggia, E. D., Smith, F. Entropic Elasticity of l-phage DNA. Science. 265 (5178), 1599-1600 (1994).
- Marko, J. F., Siggia, E. D. Stretching DNA. Macromolecules. 28 (26), 8759-8770 (1995).
- Puchner, E. M., Franzen, G., Gautel, M., Gaub, H. E. Comparing Proteins by Their Unfolding Pattern. Biophys. J. 95 (1), 426-434 (2008).
- Livadaru, L., Netz, R. R., Kreuzer, H. J. Stretching Response of Discrete Semiflexible Polymers. Macromolecules. 36 (10), 3732-3744 (2003).
- Zimmermann, J. L., Nicolaus, T., Neuert, G., Blank, K. Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments. Nat. Protoc. 5 (6), 975-985 (2010).
- Gumpp, H., Stahl, S. W., Strackharn, M., Puchner, E. M., Gaub, H. E. Ultrastable combined atomic force and total internal fluorescence microscope. Rev. Sci. Instrum. 80 (6), 063704 (2009).
- Gustafsson, M. G. L., Clarke, J. Scanning force microscope springs optimized for optical-beam deflection and with tips made by controlled fracture. J. Appl. Phys. 76 (1), 172 (1994).
- Cook, S. M., Lang, K. M., Chynoweth, K. M., Wigton, M., Simmonds, R. W., Schäffer, T. E. Practical implementation of dynamic methods for measuring atomic force microscope cantilever spring constants. Nanotechnology. 17 (9), 2135-2145 (2006).
- Proksch, R., Schäffer, T. E., Cleveland, J. P., Callahan, R. C., Viani, M. B. Finite optical spot size and position corrections in thermal spring constant calibration. Nanotechnology. 15 (9), 1344-1350 (2004).
- Ainavarapu, S. R. K., Brujic, J., et al. Contour Length and Refolding Rate of a Small Protein Controlled by Engineered Disulfide Bonds. Biophys. J. 92 (1), 225-233 (2007).
- Dietz, H., Rief, M. Detecting Molecular Fingerprints in Single Molecule Force Spectroscopy Using Pattern Recognition. Jap. J. Appl. Phys. 46, 5540-5542 (2007).
- Evans, E., Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 72 (4), 1541-1555 (1997).
- Dietz, H., Rief, M. Protein structure by mechanical triangulation. Proc. Natl. Acad. Sci. U.S.A. 103 (5), 1244-1247 (2006).
- Dudko, O. K., Hummer, G., Szabo, A. Intrinsic Rates and Activation Free Energies from Single-Molecule Pulling Experiments. Phys. Rev. Lett. 96 (10), 108101 (2006).
- Friddle, R. W., Noy, A., De Yoreo, J. J. Interpreting the widespread nonlinear force spectra of intermolecular bonds. Proc. Natl. Acad. Sci. U.S.A. 109 (34), 13573-13578 (2012).
