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A single‐molecule fluorescence system for studying adenylate kinase under force
Author(s) -
Wilson Christian A.M.,
Leachman Samuel M.,
Cervantes Bernardo,
Ierokomos Athena,
Marqusee Susan,
Bustamante Carlos
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.996.10
Subject(s) - magnetic tweezers , total internal reflection fluorescence microscope , förster resonance energy transfer , chemistry , fluorescence , force spectroscopy , biophysics , molecule , optical tweezers , streptavidin , fluorescence microscope , macromolecule , nanotechnology , crystallography , biochemistry , biotin , materials science , optics , physics , organic chemistry , biology , membrane
Single‐molecule manipulation has increasingly become a useful method for studying macromolecular dynamics. We have built a novel instrument for force spectroscopy that combines the capabilities of magnetic tweezers and single molecule Förster resonance energy transfer (smFRET). A supermagnet exerts force on 2.1 μm antidigoxigenin‐coated paramagnetic beads tethered to specific proteins under study. These fluorescently labeled proteins are functionalized with DNA handles containing biotin and immobilized on the surface of a flow chamber in a total internal reflection fluorescent (TIRF) microscope via streptavidin interactions. The distance between dyes from each molecule that is attached to a bead in the microscope's field of view can be monitored as a function of force. The model system that we are currently studying is adenylate kinase (AK). This enzyme was successfully labeled with fluorescent dyes and DNA using click chemistry and cysteine chemistry. AK was first characterized in optical tweezers and was found to unfold around 25 pN during force‐extension experiments with a fast 4 nm intermediate at 15 pN. This intermediate could correspond to the ATP binding domain unfolding independently of the rest of the protein. Preliminary fluorescence data from our instrument confirms the existence of this intermediate under force. HHMI‐CB; NIH‐SM