title: Nanomechanics of HaloTag tethers.
abstract: The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ∼2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.
authors.affiliation: Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, New York 10027, USA. ivp2105@columbia.edu
authors.forename: Ionel
authors.initials: I
authors.lastname: Popa
authors.forename: Ronen
authors.initials: R
authors.lastname: Berkovich
authors.forename: Jorge
authors.initials: J
authors.lastname: Alegre-Cebollada
authors.forename: Carmen L
authors.initials: CL
authors.lastname: Badilla
authors.forename: Jaime Andrés
authors.initials: JA
authors.lastname: Rivas-Pardo
authors.forename: Yukinori
authors.initials: Y
authors.lastname: Taniguchi
authors.forename: Masaru
authors.initials: M
authors.lastname: Kawakami
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernandez
mesh_terms: D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D055595:Mechanical Phenomena; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D036103:Nanotechnology; D017510:Protein Folding
chemical_list: D006867:Hydrolases; C046137:haloalkane dehalogenase
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