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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