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 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 four-fold increase, from 131 pN 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 more 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.

Quantifying thiol–gold interactions towards the efficient strength control

“…The quantitative information obtained on the stability of isolated thiol-gold contacts by using AFM-based SMFS is useful for sample systems where the strength of individual thiol-gold contact plays important roles, such as the picking up (or immobilization) and manipulation of thiol-labelled polyethylene oxide chain (or polyprotein) 22,59,60 . An in situ waiting time of Z3.0 s for the formation of covalent S-Au bond is necessary to stabilize the thiol-gold chemistry-based molecular linkages.…”

“…For example, to stabilize the anchor of single thiol-labelled molecules, we can use an oxidized gold surface and perform the reaction in aqueous (rather than in ethanolic) solution at higher pH with appropriate reaction time (for example, B3.0 s for isolated thiol-gold contacts, while less than 1 day for SAMs). For the single-molecule pulling experiment, in which a thiol-labelled molecule needs to be attached to the gold substrate and the strength of the thiol-gold contact is crucial to the experiment 18,60 , the molecule of interest needs to be immobilized onto the gold surface under a more dilute solution; the utilization of small thiol molecules as a 'dilute agent' is also avoided to co-assemble the target molecule 13 .…”

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

“…Moreover, the attachment of pili to the surface and cantilever relies on nonspecific interactions, such that the vector of the force is not explicitly defined. To overcome these limitations, we produced an engineered heteropolyprotein of the SpaA pilin with a C-terminal Cys for covalent attachment to gold-coated cantilevers, along with an N-terminal HaloTag protein that allows covalent tethering to functionalized glass surfaces (22,23) (Fig. 3A).…”

“…3A). Such covalent anchorage ensures single-molecule tethers that can withstand forces close to 1 nN (23). Importantly, SpaA is pulled with the same geometry in the heteropolyprotein as in native pili.…”

“…We synthesized a pilus-like FimA polyprotein construct of four FimA pilins in series, along with an N-terminal HaloTag and a C-terminal Cys for covalent tethering (23) (Fig. 3D).…”