Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high‐precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin. A rapid near‐equilibrium folding between the first three unfolding states was discovered, the two transitions corresponded to the unfolding of five and three amino acids, respectively, when using a cantilever optimized for 2 μs resolution. The third of these states was retinal‐stabilized and previously undetected, despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for more than 1 min. This ability to measure the dynamics of the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native‐like conditions.     

Introduction of steered molecular dynamics into UNRES coarse‐grained simulations package

In this article, an implementation of steered molecular dynamics (SMD) in coarse‐grain UNited RESidue (UNRES) simulations package is presented. Two variants of SMD have been implemented: with a constant force and a constant velocity. The huge advantage of SMD implementation in the UNRES force field is that it allows to pull with the speed significantly lower than the accessible pulling speed in simulations with all‐atom representation of a system, with respect to a reasonable computational time. Therefore, obtaining pulling speed closer to those which appear in the atomic force spectroscopy is possible. The newly implemented method has been tested for behavior in a microcanonical run to verify the influence of introduction of artificial constrains on keeping total energy of the system. Moreover, as time dependent artificial force was introduced, the thermostat behavior was tested. The new method was also tested via unfolding of the Fn3 domain of human contactin 1 protein and the I27 titin domain. Obtained results were compared with Gø‐like force field, all‐atom force field, and experimental results. © 2017 Wiley Periodicals, Inc.     

A Single‐Molecule Perspective on the Role of Solvent Hydrogen Bonds in Protein Folding and Chemical Reactions

We present an array of force spectroscopy experiments that aim to identify the role of solvent hydrogen bonds in protein folding and chemical reactions at the single‐molecule level. In our experiments we control the strength of hydrogen bonds in the solvent environment by substituting water (H₂O) with deuterium oxide (D₂O). Using a combination of force protocols, we demonstrate that protein unfolding, protein collapse, protein folding and a chemical reaction are affected in different ways by substituting H₂O with D₂O. We find that D₂O molecules form an integral part of the unfolding transition structure of the immunoglobulin module of human cardiac titin, I27. Strikingly, we find that D₂O is a worse solvent than H₂O for the protein I27, in direct contrast with the behaviour of simple hydrocarbons. We measure the effect of substituting H₂O with D₂O on the force dependent rate of reduction of a disulphide bond engineered within a single protein. Altogether, these experiments provide new information on the nature of the underlying interactions in protein folding and chemical reactions and demonstrate the power of single‐molecule techniques to identify the changes induced by a small change in hydrogen bond strength.     

Simulation of the mechanical unfolding of ubiquitin: probing different unfolding reaction coordinates by changing the pulling geometry

Motivated by the recent experimental atomic force microscopy (AFM) measurements of the mechanical unfolding of proteins pulled in different directions [D. J. Brockwell et al., Nat. Struct. Biol. 10, 731 (2003); M. Carrion-Vazquez et al., ibid 10, 738 (2003)] we have computed the unfolding free energy profiles for the ubiquitin domain when it is stretched between its (A) N and C termini, (B) Lys48 and C terminus, (C) Lys11 and C terminus, and (D) N terminus and Lys63. Our results for cases (A) and (B) are in good agreement with the experimental unfolding forces measured for the N-C and Lys48-C linked polyubiquitin, in particular, indicating a considerably lower unfolding force in the latter case. Mechanical unfolding in case (A) involves longitudinal shearing of the terminal parallel strands while in case (C) the same strands are "unzipped" by the force. The computed unfolding forces in case (C) are found to be very low, less than 50 pN for pulling rates typical of AFM experiments. The unfolding free energy barrier found in case (C) is approximately 13 kcal/mol, which corresponds to a zero-force unfolding rate constant that is comparable to the rate of chemical unfolding extrapolated to zero denaturant concentration. The unfolding barrier calculated in case (A) in the limit of zero force is much higher, suggesting that mechanical unfolding in this case follows a pathway that is different from that of thermal/chemical denaturation.     

Effects of contact force and salt concentration on the unbinding of a DNA duplex by force spectroscopy

We report that varying the contact force in force spectroscopy results in a significant shift in DNA unbinding forces, measured from short oligonucleotides using a PicoForce microscope. The contact force between a 30-mer complementary DNA-coated probe and surface was varied from 100 pN to 10 nN, resulting in a significant shift in the most abundant unbinding force measured between the duplex. When contact forces were set at 200 pN or less, which is generally considered to be a low contact force region for biomolecular force spectroscopy studies, the shift in DNA unbinding forces was significant with changes in contact force. The effect of the salt concentration on the DNA unbinding forces was also examined for a range of salt concentrations from 5 to 500 mM because the presence of salt ions is necessary to facilitate the hybridization process. Although an increase in salt concentration resulted in the facilitation of DNA multiple binding events during force spectroscopy measurements, no significant shift in unbinding forces was observed. Our experiment demonstrates that the wide variation in DNA unbinding forces reported in the literature (50-600 pN) for short oligonucleotides can be accounted for by the different contact forces used and shows little or no effect of the salt concentration used in those studies. Furthermore, this study demonstrates the importance of reporting contact forces in force spectroscopy measurements for quantitative comparisons between different biomolecular systems, especially for noncovalent-type interactions.     

Synthesis and single-molecule studies of a well-defined biomimetic modular multidomain polymer using a peptidomimetic beta-sheet module

In the pursuit of advanced biomaterials with combined strength, toughness, and elasticity, a new class of well-defined modular polymers has been synthesized, and their nanomechanical properties have been studied using atomic force microscopy. These polymers are based on a peptidomimetic beta-sheet-based double-closed loop (DCL) module, which was designed to overcome the limitation of the modular polymers we reported previously (J. Am. Chem. Soc. 2004, 126, 2059). Single-molecule force-extension experiments revealed the sequential unfolding of these modules as the polymer is stretched, resulting in more regular sawtooth-patterned curves similar to those seen in titin and other biopolymers. The single-molecule data agreed well with computer modeling, which suggested that hydrogen bonding and pi-stacking are both involved in the formation of small DCL clusters along the polymer chain.     

Intermolecular forces between acetylcholine and acetylcholinesterases studied with atomic force microscopy

With the aid of atomic force microscopy, the intermolecular forces between acetyleholinesterases (AChE) and its natural substrate acetylcholine (ACh) have been studied. Through force spectrum measurement based on imaging of AChE molecules it was found that the attraction force between individual molecule pairs of ACh and AChE was (10±1) pN just before the quaternary ammonium head of ACh got into contact with the negative end of AChE and the decaying distance of attraction was (4±1) nm from the surface of ACHE. The adhesion force between individual ACh and AChE molecule pairs was (25±2) pN, which had a decaying feature of fast-slow-fast (FSF). The attraction forces between AChE and choline (Ch), the quaternary ammonium moiety and hydrolysate of ACh molecule, were similar to those between AChE and ACh. The adhesion forces between AChE and Ch were (20±2) pN, a little weaker than that between ACh and ACHE. These results indicated that AChE had a steering role for the diffusion of ACh toward it and had r     

Evidence of Splitting 1,2,3‐Triazole into an Alkyne and Azide by Low Mechanical Force in the Presence of Other Covalent Bonds

The cycloaddition reaction of an alkyne and azide to form a 1,2,3‐triazole is widely used in many areas. However, the stability of the triazole moiety under mechanical stress is unclear. To see if a triazole could be selectively split into an alkyne and azide in the presence of other typical covalent bonds, a mica surface functionalized with a molecule containing a triazole moiety in the middle and an activated ester at the end was prepared. An atomic force microscope (AFM) tip with amino groups on its surface was ramped over the mica surface at predefined locations, which could temporarily link the tip to the surface through amide bond formation. During retraction, the triazole or another bond in the linkage broke, and a force was recorded. The forces varied widely at different ramps from close to 0 pN to 860 pN due to nonspecific adhesions and to the inherent inconsistency of single bond rupture. If some of the forces were from triazole cycloreversion, there would be alkynes at the predefined ramping locations. The surface was reacted with an azide carboxylic acid followed by labeling with amino Au nanoparticles (AuNPs). AFM imaging revealed AuNPs at the predicted locations, which provided evidence that under certain conditions triazole could be split selectively in the presence of other bonds at forces below 860 pN.     

Phosphorylation Reduces the Mechanical Stability of the α‐Catenin/ β‐Catenin Complex

The α‐catenin/β‐catenin complex serves as a critical molecular interface involved in cadherin–catenin‐based mechanosensing at the cell–cell adherence junction that plays a critical role in tissue integrity, repair, and embryonic development. This complex is subject to tensile forces due to internal actomyosin contractility and external mechanical micro‐environmental perturbation. However, the mechanical stability of this complex has yet to be quantified. Here, we directly quantified the mechanical stability of the α‐catenin/β‐catenin complex and showed that it has enough mechanical stability to survive for tens to hundreds of seconds within physiological level of forces up to 10 pN. Phosphorylation or phosphotyrosine‐mimetic mutations (Y142E or/and T120E) on β‐catenin shorten the mechanical lifetime of the complex by tens of fold over the same force range. These results provide insights into the regulation of the α‐catenin/β‐catenin complex by phosphorylation.     

Mechanical Activation Drastically Accelerates Amide Bond Hydrolysis,Matching Enzyme Activity

Amide bonds, which include peptide bonds connecting amino acids in proteins and polypeptides, give proteins and synthetic polyamides their enormous strength. Although proteins and polyamides sustain mechanical force in nature and technology, how forces affect amide and peptide bond stability is still unknown. Using single‐molecule force spectroscopy, we discover that forces of only a few hundred pN accelerate amide hydrolysis 10⁹‐fold, an acceleration hitherto only known from proteolytic enzymes. The drastic acceleration at low force precedes a moderate additional acceleration at nN forces. Quantum mechanochemical ab initio calculations explain these experimental results mechanistically and kinetically. Our findings reveal that, in contrast to previous belief, amide stability is strongly force dependent. These calculations provide a fundamental understanding of the role of mechanical activation in amide hydrolysis and point the way to potential applications from the recycling of macromolecular waste to the design of bioengineered proteolytic enzymes.     

Atomistic evidence of how force dynamically regulates thiol/disulfide exchange

The intricate coupling of mechanical force and chemical reactivity has been increasingly revealed in recent years by force spectroscopy experiments on the thiol/disulfide exchange reaction. We here aimed at elucidating the underlying dynamic effects of force on the reaction center for the case of disulfide bond reduction by dithiothreitol at forces of 200-2000 pN, by combining transition path sampling and quantum/classical mechanical simulations. Reaction rates and their dependence on force as quantified by Δx(r), the distance between reactant and transition state, are in good agreement with experiments but indicate a shift of the transition state structure at high forces. Indeed, while an associate S(N)2 mechanism prevails, force causes a move of the transition state to a longer length of the cleaving bond and a shorter length of the forming disulfide bond. Our results highlight the distribution of force into various degrees of freedom, which implies that care must be taken when correlating Δx(r) with a single order parameter of the reaction.     

Direct detection by atomic force microscopy of single bond forces associated with the rupture of discrete charge-transfer complexes

Atomic force microscopy (AFM) was used to measure the chemical binding force of discrete electron donor-acceptor complexes formed at the interface between proximal self-assembled monolayers (SAMs). Derivatives of the well-known electron donor N,N,N',N'-tetramethylphenylenediamine (TMPD) and the electron acceptor 7,7,8,8-tetracyanoquinodimethane (TCNQ) were immobilized on Au-coated AFM tips and substrates by formation of SAMs of N,N,N'-trimethyl-N'-(10-thiodecyl)-1,4-phenylenediamine (I) and bis(10-(2-((2,5-cyclohexadiene-1,4-diylidene)dimalonitrile))decyl) disulfide (II), respectively. Pull-off forces between modified tips and substrates were measured under CHCl(3) solvent. The mean pull-off forces associated with TMPD/TCNQ microcontacts were more than an order of magnitude larger than the pull-off forces for TMPD/TMPD and TCNQ/TCNQ microcontacts, consistent with the presence of specific charge-transfer interactions between proximal TMPD donors and TCNQ acceptors. Furthermore, histograms of pull-off forces for TMPD/TCNQ contacts displayed 70 +/- 15 pN periodicity, assigned to the rupture of individual TMPD-TCNQ donor-acceptor (charge-transfer) complexes. Both the mean pull-off force and the 70 pN force quantum compare favorably with a contact mechanics model that incorporates the effects of discrete chemical bonds, solvent surface tensions, and random contact area variations in consecutive pull-offs. From the 70 pN force quantum, we estimate the single bond energy to be approximately 4-5 kJ/mol, in reasonable agreement with thermodynamic data. These experiments establish that binding forces due to discrete chemical bonds can be detected directly in AFM pull-off measurements employing SAM modified probes and substrates. Because SAMs can be prepared with a wide range of exposed functional groups, pull-off measurements between SAM-coated tips and substrates may provide a general strategy for directly measuring binding forces associated with a variety of simple, discrete chemical bonds, e.g., single hydrogen bonds.     

Topography of the free-energy landscape probed via mechanical unfolding of proteins

Single-molecule experiments in which proteins are unfolded by applying mechanical stretching forces generally force unfolding to proceed along a reaction coordinate that is different from that in chemical or thermal denaturation. Here we simulate the mechanical unfolding and refolding of a minimalist off-lattice model of the protein ubiquitin to explore in detail the slice of the multidimensional free-energy landscape that is accessible via mechanical pulling experiments. We find that while the free-energy profile along typical "chemical" reaction coordinates may exhibit two minima, corresponding to the native and denatured states, the free energy G(z) is typically a monotonic function of the mechanical coordinate z equal to the protein extension. Application of a stretching force along z tilts the free-energy landscape resulting in a bistable (or multistable) free energy G(z)-fz probed in mechanical unfolding experiments. We construct a two-dimensional free-energy surface as a function of both chemical and mechanical reaction coordinates and examine the coupling between the two. We further study the refolding trajectories after the protein has been prestretched by a large force, as well as the mechanical unfolding trajectories in the presence of a large stretching force. We demonstrate that the stretching forces required to destabilize the native state thermodynamically are larger than those expected on the basis of previous experimental estimates of G(z). This finding is consistent with the recent experimental studies, indicating that proteins may refold even in the presence of a substantial stretching force. Finally, we show that for certain temperatures the free energy of a polyprotein chain consisting of multiple domains is a linear function of the chain extension. We propose that the recently observed "slow phase" in the refolding of proteins under mechanical tension may be viewed as downhill diffusion in such a linear potential.     

Compaction dynamics of single DNA molecules under tension

Using transverse magnetic tweezers, we studied the dynamics of DNA compaction induced by hexaammine cobalt chloride under constant forces. Discontinuous DNA compaction events were found for forces ranging from 0.5 to 1.7 pN, with approximately 270 nm DNA adsorbed in each compaction event. Forces larger than 6 pN were found able to unravel the toroid in a similar intermittent stepwise manner. The observations indicate that the folding/unfolding events are transitions between two metastable structural states which are separated by a tension-dependent energy barrier. Analysis of the waiting time revealed that the degree of the package ordering of DNA in a toroid depends on the compaction kinetics.     

Multivalent Interactions of Polyamide Based Sequence‐Controlled Glycomacromolecules with Concanavalin A

Binding of mannose presenting macromolecules to the protein receptor concanavalin A (ConA) is investigated by means of single‐molecule atomic force spectroscopy (SMFS) in combination with dynamic light scattering and molecular modeling. Oligomeric (M_w ≈ 1.5–2.5 kDa) and polymeric (M_w ≈ 22–30 kDa) glycomacromolecules with controlled number and positioning of mannose units along the scaffolds accessible by combining solid phase synthesis and thiol–ene coupling are used as model systems to assess the molecular mechanisms that contribute to multivalent ConA–mannose complexes. SMFS measurements show increasing dissociation force from monovalent (≈57 pN) to pentavalent oligomers (≈75 pN) suggesting subsite binding to ConA. Polymeric glycomacromolecules with larger hydrodynamic diameters compared to the binding site spacing of ConA exhibit larger dissociation forces (≈80 pN), indicating simultaneous dissociation from multiple ConA binding sites. Nevertheless, although simultaneous dissociation of multiple ligands could be expected for such multivalent systems, predominantly single dissociation events are observed. This is rationalized by strong coiling of the macromolecules' polyamide backbone due to intramolecular hydrogen bonding hindering unfolding of the coil. Therefore, this study shows that the design of glycopolymers for multivalent receptor binding and clustering must consider 3D structure and intramolecular interactions of the scaffold.     

Mg2+‐Dependent High Mechanical Anisotropy of Three‐Way‐Junction pRNA as Revealed by Single‐Molecule Force Spectroscopy

Mechanical anisotropy is ubiquitous in biological tissues but is hard to reproduce in synthetic biomaterials. Developing molecular building blocks with anisotropic mechanical response is the key towards engineering anisotropic biomaterials. The three‐way‐junction (3WJ) pRNA, derived from ϕ 29 DNA packaging motor, shows strong mechanical anisotropy upon Mg²⁺ binding. In the absence of Mg²⁺, 3WJ‐pRNA is mechanically weak without noticeable mechanical anisotropy. In the presence of Mg²⁺, the unfolding forces can differ by more than 4‐fold along different pulling directions, ranging from about 47 pN to about 219 pN. Mechanical anisotropy of 3WJ‐pRNA stems from pulling direction dependent cooperativity for the rupture of two Mg²⁺ binding sites, which is a novel mechanism for the mechanical anisotropy of biomacromolecules. It is anticipated that 3WJ‐pRNA can be used as a key element for the construction of biomaterials with controllable mechanical anisotropy.     

Single Molecule Force Spectroscopy Reveals Critical Roles of Hydrophobic Core Packing in Determining the Mechanical Stability of Protein GB1

Understanding molecular determinants of protein mechanical stability is important not only for elucidating how elastomeric proteins are designed and functioning in biological systems but also for designing protein building blocks with defined nanomechanical properties for constructing novel biomaterials. GB1 is a small α/β protein and exhibits significant mechanical stability. It is thought that the shear topology of GB1 plays an important role in determining its mechanical stability. Here, we combine single molecule atomic force microscopy and protein engineering techniques to investigate the effect of side chain reduction and hydrophobic core packing on the mechanical stability of GB1. We engineered seven point mutants and carried out mechanical ?-value analysis of the mechanical unfolding of GB1. We found that three mutations, which are across the surfaces of two subdomains that are to be sheared by the applied stretching force, in the hydrophobic core (F30L, Y45L, and F52L) result in significant decrease in mechanical unfolding force of GB1. The mechanical unfolding force of these mutants drop by 50-90 pN compared with wild-type GB1, which unfolds at around 180 pN at a pulling speed of 400 nm/s. These results indicate that hydrophobic core packing plays an important role in determining the mechanical stability of GB1 and suggest that optimizing hydrophobic interactions across the surfaces that are to be sheared will likely be an efficient method to enhance the mechanical stability of GB1 and GB1 homologues.     

Mechanical unfolding of ensemble biomolecular structures by shear force

Mechanical unfolding of biomolecular structures has been exclusively performed at the single-molecule level by single-molecule force spectroscopy (SMFS) techniques. Here we transformed sophisticated mechanical investigations on individual molecules into a simple platform suitable for molecular ensembles. By using shear flow inside a homogenizer tip, DNA secondary structures such as i-motifs are unfolded by shear force up to 50 pN at a 77 796 s⁻¹ shear rate. We found that the larger the molecules, the higher the exerted shear forces. This shear force approach revealed affinity between ligands and i-motif structures. It also demonstrated a mechano-click reaction in which a Cu(i) catalyzed azide–alkyne cycloaddition was modulated by shear force. We anticipate that this ensemble force spectroscopy method can investigate intra- and inter-molecular interactions with the throughput, accuracy, and robustness unparalleled to those of SMFS methods.

Shear force in a homogenizer mechanically unfolds an ensemble set of biomolecular structures.     

Adhesion of single polyelectrolyte molecules on silica, mica, and bitumen surfaces

In a recent study (Energy Fuels 2005, 19, 936), a partially hydrolyzed polyacrylamide (HPAM) was used as a process aid to recover bitumen from oil sand ores. It was found that HPAM addition at the bitumen extraction step not only improved bitumen recovery but also enhanced fine solids settling in the tailings stream. To understand the role of HPAM, single-molecule force spectroscopy was employed for the first time to measure the desorption/adhesion forces of single HPAM molecules on silica, mica, and bitumen surfaces using an atomic force microscope (AFM). Silicon wafers with an oxidized surface layer and newly cleaved mica were used, respectively, to represent sand grains and clays in oil sands. The force measurements were carried out in deionized water and in commercial plant process water under equilibrium conditions. The desorption/adhesion forces of HPAM obtained on mica, silica, and bitumen surfaces were approximately 200, 40, and 80 pN in deionized water and approximately 100, 50, and 40 pN in the plant process water, respectively. The measured adhesion forces together with the zeta potential values of these surfaces indicate that the polymer would preferentially adsorb onto clay surfaces rather than onto bitumen surfaces. It is the selective adsorption of HPAM that benefits both bitumen recovery and tailings settling when the polymer was added directly to the bitumen extraction process at an appropriate dosage.