title: A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics.
abstract: Under physiological conditions, protein oxidation and misfolding occur with very low probability and on long times scales. Single-molecule techniques provide the ability to distinguish between properly folded and damaged proteins that are otherwise masked in ensemble measurements. However, at physiological conditions these rare events occur with a time constant of several hours, inaccessible to current single-molecule approaches. Here we present a magnetic-tweezers-based technique that allows, for the first time, the study of folding of single proteins during week-long experiments. This technique combines HaloTag anchoring, sub-micrometer positioning of magnets, and an active correction of the focal drift. Using this technique and protein L as a molecular template, we generate a magnet law by correlating the distance between the magnet and the measuring paramagnetic bead with unfolding/folding steps. We demonstrate that, using this magnet law, we can accurately measure the dynamics of proteins over a wide range of forces, with minimal dispersion from bead to bead. We also show that the force calibration remains invariant over week-long experiments applied to the same single proteins. The approach demonstrated in this Article opens new, exciting ways to examine proteins on the "human" time scale and establishes magnetic tweezers as a valuable technique to study low-probability events that occur during protein folding under force.
authors.affiliation: Department of Biological Sciences, Columbia University , 1212 Amsterdam Avenue, New York, New York 10027, United States.
authors.forename: Ionel
authors.initials: I
authors.lastname: Popa
authors.affiliation: Department of Biological Sciences, Columbia University , 1212 Amsterdam Avenue, New York, New York 10027, United States.
authors.forename: Jaime Andrés
authors.initials: JA
authors.lastname: Rivas-Pardo
authors.affiliation: Department of Biological Sciences, Columbia University , 1212 Amsterdam Avenue, New York, New York 10027, United States.
authors.forename: Edward C
authors.initials: EC
authors.lastname: Eckels
authors.affiliation: Department of Biological Sciences, Columbia University , 1212 Amsterdam Avenue, New York, New York 10027, United States.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Echelman
authors.affiliation: Department of Biological Sciences, Columbia University , 1212 Amsterdam Avenue, New York, New York 10027, United States.
authors.forename: Carmen L
authors.initials: CL
authors.lastname: Badilla
authors.affiliation: Department of Biological Sciences, Columbia University , 1212 Amsterdam Avenue, New York, New York 10027, United States.
authors.forename: Jessica
authors.initials: J
authors.lastname: Valle-Orero
authors.affiliation: Department of Biological Sciences, Columbia University , 1212 Amsterdam Avenue, New York, New York 10027, United States.
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernández
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D059346:Magnets; D055595:Mechanical Phenomena; D017510:Protein Folding
chemical_list: D001426:Bacterial Proteins