Nanomechanics of HaloTag tethers.
J Am Chem Soc
; 135(34): 12762-71, 2013 Aug 28.
Article
en En
| MEDLINE
| ID: mdl-23909704
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 â¼2000 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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Nanotecnología
/
Hidrolasas
Idioma:
En
Revista:
J Am Chem Soc
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos