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Bioengineering

AFM-tabanlı Tek-molekül Kuvvetleri Spektroskopisi ile cellulosome Reseptör-ligand Sistemleri Araştırılması

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

Abstract

Cellulosomes lignoselülozik alt tabakaların sindirimi anaerobik bakteri ve mantar bir alt kümesi tarafından kullanılan ayrı çoklu enzim kompleksleridir. Katalitik olmayan iskele proteine ​​enzimlerin montaj etkileşim cohesin ve dockerin modülleri içeren ilgili reseptör-ligand çifti bir aile arasındaki etkileşimler ile yönlendirilir. Konvansiyonel dökme yöntemleri ile doğru off-rate ölçümlerini engel olabilir nanomolar aralığında düşük pikomolar DİSOSİASYON sabitler, içinde ve kohezinin dockerin modüller sonuçlar arasında son derece güçlü bağlama. Atomik kuvvet mikroskopu ile tek molekül kuvvet spektroskopisi (SMFs) zorlamak için bireysel biyomoleküllerin tepki ölçer ve diğer tek molekül manipülasyon yöntemleri (örneğin optik cımbız) aksine, yüksek afiniteli reseptör-ligand etkileşimlerini incelemek için uygun olduğu için yüksek kuvvet rejimi (> 120 pN) prob kabiliyetini. Burada Cellul eğitim için tam bizim protokol mevcuttek-molekül düzeyinde protein osomal meclisleri. Yerel cellulosome türetilen bir protein topoloji kullanarak, modül cohesin (CBM-cohesin) füzyon proteinleri, bir sistein artığı da erişilebilir bir serbest tiyol grubu ile her bir bağlama enzim dockerin ve karbohidrat ile çalışmıştır. Biz yüksek yakınlık kompleksi için ayrıntılı bağlama parametrelerini elde etmek için bizim ölçüm ve veri analizi prosedürü ile birlikte, sitemiz-spesifik yüzey immobilizasyon protokol mevcut. Biz tek subdomain açılımı güçleri, karmaşık kopma kuvvetleri, off-oranları kinetik ve iyi bağlayıcı potansiyel genişlikleri ölçmek nasıl gösterilmektedir. Multidomain selülolitik komplekslerinin topluluğundan sorumlu-cohesin dockerin etkileşimi karakterize etmede, bu yöntemlerin başarılı bir uygulama daha da tarif edilmektedir.

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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AFM-tabanlı Tek-molekül Kuvvetleri Spektroskopisi ile cellulosome Reseptör-ligand Sistemleri Araştırılması
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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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