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Manipulating and Detecting a Protein's Energy Landscape
Author(s) -
Marqusee Susan
Publication year - 2008
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.22.1_supplement.407.3
Subject(s) - optical tweezers , energy landscape , pipette , bead , molecule , chemical physics , laser , spheres , chemistry , nanotechnology , anisotropy , materials science , biophysics , optics , physics , composite material , biochemistry , organic chemistry , astronomy , biology
We have developed a novel method to explore the single molecule behavior of proteins in the low force regime of the laser tweezers. Individual protein molecules are tethered between two polystyrene beads using two dsDNA molecules, which are in turn attached to the protein through unique engineered cysteine residues. One bead is held by suction on the tip of a pipette while the other is held in the optical trap. The force and distance between the beads is varied by moving the bead on the pipette relative to the bead in the trap. The DNA handles can be attached to any exposed cysteine residues, allowing a variety of pulling geometries to be used to investigate the anisotropy of the protein energy landscape. The low spring of laser tweezers enabled us to directly monitor low force refolding events and fluctuations between different molecular structures at quasi‐equilibrium conditions. Data exploring the behavior of different protein protein topologies, different pulling geometries and the relationship to bulk solution studies of the energy landscape will be presented.