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Bioengineering

Gransker Receptor-ligandsysternene med Cellulosome med AFM-baserte Single-molekyl kraft spektroskopi

doi: 10.3791/50950 Published: December 20, 2013
* These authors contributed equally

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.

Materials

Name Company Catalog Number Comments
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

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Gransker Receptor-ligandsysternene med Cellulosome med AFM-baserte Single-molekyl kraft spektroskopi
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Cite this Article

Jobst, M. A., Schoeler, C., Malinowska, K., Nash, M. A. Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy. J. Vis. Exp. (82), e50950, doi:10.3791/50950 (2013).More

Jobst, M. A., Schoeler, C., Malinowska, K., Nash, M. A. Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy. J. Vis. Exp. (82), e50950, doi:10.3791/50950 (2013).

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