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.

a new active dimension of "time" has evolved, leading to the new concept of 4D printing, which refers to the ability of 3D printed structures to actively transform over time in response to environmental stimuli. [5] Smart or stimuliresponsive materials have the unique ability to return from a temporary deformed state, induced by heat, light, pH, ultrasound, chemical substances, [6][7][8][9][10][11][12][13] etc., to their permanent, i.e., original, shape, thus exhibiting advantages for applications in numerous sectors, such as sensors and actuators, [14] tissue engineering, [15] bio-separation devices, and controlled drug delivery. [16][17][18][19][20][21] To date, two main types of materials have been considered to realize 4D printing: shape memory polymers (SMPs) and hydrogels. SMP-based 4D printing offers structural modification and recovery in response to temperature, which are established through complex functionalities of multiple or reversible shape switching, and such printing may provide inspiration for the molecular architecture of shape memory hydrogels (SMHs). However, SMPs cannot completely replace hydrophilic soft materials due to the limitations arising from their sustainability in wet environments, rigidity, material permeability, and biological compatibility. [22] Therefore, mechanically active, self-shaping hydrogels that undergo desired, programmable 3D shape transformations and execute mechanical tasks as soft robots under an external trigger have recently attracted growing interest. The use of a hydrogel system in soft robotic counterparts offers distinct advantages: simple designs, low cost, processability at low temperatures and in aqueous environments, and the possibility to mimic human functionality. [23,24] Directed movement of hydrogels can be obtained by expansion/contraction, for example, by isotropic volume expansion or shrinkage of homogeneous hydrogels or by the bending/unbending approach, which represents an anisotropic deformation and often involves fabrication of a hydrogel structure with two layers with different swellability values. [25][26][27][28][29] The first hydrogel-based bilayer actuation system composed of pNIPAM and acrylamide, obtained through conventional mold techniques, was demonstrated by Hu et al.; [25] after that, a range of self-assembled, origami-inspired structures were reported, [27,[30][31][32][33][34][35][36][37][38] but only a few works successfully realized 4D printing with hydrogels. [28,29] Some noteworthy Hydrogel actuators with soft-robotic functions and biomimetic advanced materials with facile and programmable fabrication processes remain scarce. A novel approach to fabricating a shape-memory-hydrogel-(SMG)-based bilayer system using 3D printing to yield a soft actuator responsive to the methodical application of swelling and heat is introduced. Each layer of the bilayer is composed of poly(N,N-dimethyl acrylamide-co-stearyl acrylate) (P(DMAAm-co-SA))-based hydrogels with different concentrations of the crystalline monomer SA within the SMG network...

The ability of proteins and their complexes to withstand or respond to mechanical stimuli is vital for cells to maintain their structural organisation, to relay external signals and to facilitate unfolding and remodelling. Force spectroscopy using the atomic force microscope allows the behaviour of single protein molecules under an applied extension to be investigated and their mechanical strength to be quantified. protein L, a simple model protein, displays moderate mechanical strength and is thought to unfold by the shearing of two mechanical sub-domains. Here, we investigate the importance of side-chain packing for the mechanical strength of protein L by measuring the mechanical strength of a series of protein L variants containing single conservative hydrophobic volume deletion mutants. Of the five thermodynamically destabilised variants characterised, only one residue (I60V) close to the interface between two mechanical sub-domains was found to differ in mechanical properties to wild type (ΔFI60V–WT = − 36 pN at 447 nm s− 1, ΔxuI60V–WT = 0.2 nm). Φ-value analysis of the unfolding data revealed a highly native transition state. To test whether the number of hydrophobic contacts across the mechanical interface does affect the mechanical strength of protein L, we measured the mechanical properties of two further variants. protein L L10F, which increases core packing but does not enhance interfacial contacts, increased mechanical strength by 13 ± 11 pN at 447 nm s− 1. By contrast, protein L I60F, which increases both core and cross-interface contacts, increased mechanical strength by 72 ± 13 pN at 447 nm s− 1. These data suggest a method by which nature can evolve a varied mechanical response from a limited number of topologies and demonstrate a generic but facile method by which the mechanical strength of proteins can be rationally modified.

Experiments that measure the viscoelasticity of single molecules from the Brownian fluctuations of an atomic force microscope (AFM) have provided a new window onto their internal dynamics in an underlying conformational landscape. Here we develop and apply these methods to examine the internal friction of unfolded polypeptide chains at high stretch. The results reveal a power law dependence of internal friction with tension (exponent 1.3 +/- 0.5) and a relaxation time approximately independent of force. To explain these results we develop a frictional worm-like chain (FWLC) model based on the Rayleigh dissipation function of a stiff chain with dynamical resistance to local bending. We analyse the dissipation rate integrated over the chain length by its Fourier components to calculate an effective tension-dependent friction constant for the end-to-end vector of the chain. The result is an internal friction that increases as a power law with tension with an exponent 3/2, consistent with experiment. Extracting the intrinsic bending friction constant of the chain it is found to be approximately 7 orders of magnitude greater than expected from solvent friction alone; a possible explanation we offer is that the underlying energy landscape for bending amino acids and/or peptide bond is rough, consistent with recent results on both proteins and polysaccharides.