| Autor: |
Christian A.M. Wilson, Susan Marqusee, Carlos Bustamante, Samuel M. Leachman |
| Rok vydání: |
2012 |
| Předmět: |
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| Zdroj: |
Biophysical Journal. 102:387a |
| ISSN: |
0006-3495 |
| Popis: |
Proteins exert and withstand mechanical force in many fundamental biological processes. Optical tweezers have become a useful research tool for applying forces to single proteins and measuring the resulting changes in extension, but many interesting processes produce changes smaller than their resolution limit. Our experimental setup skirts this limitation by measuring distance changes using single-molecule Forster resonance energy transfer (smFRET) produced from a total internal reflection fluorescence (TIRF) microscope incorporating magnetic tweezers. Individual protein molecules are conjugated to FRET-paired fluorescent dyes and functionalized DNA handles using maleimide and click chemistry. These handles tether each molecule between a glass coverslip within the TIRF microscope and a paramagnetic bead. An external magnet applies a uniform field that exerts a force on each molecule tethered to the surface. Because the FRET from each molecule in the microscope's field of view can be measured simultaneously, the extension between dyes of many individual molecules as a function of force can be monitored in parallel. Here we present our initial studies using this new setup. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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