Single-molecule Mechanical Experiments Research Articles

Active unfolding of the glucocorticoid receptor by the Hsp70/Hsp40 chaperone system in single-molecule mechanical experiments

The glucocorticoid receptor (GR) is an important transcription factor and drug target linked to a variety of biological functions and diseases. It is one of the most stringent physiological clients of the Hsp90/Hsp70/Hsp40 chaperone system. In this study, we used single-molecule force spectroscopy by optical tweezers to observe the interaction of the GR’s ligand-binding domain (GR-LBD) with the Hsp70/Hsp40 chaperone system (Hsp70/40). We show in real time that Hsp70/40 can unfold the complete GR-LBD in a stepwise manner. Each unfolding step involves binding of an Hsp70 to the GR-LBD and subsequent adenosine triphosphate (ATP) hydrolysis, stimulated by Hsp40. The kinetics of chaperone-mediated unfolding depend on chaperone concentrations as well as the presence of the nucleotide exchange factor BAG1. We find that Hsp70/40 can stabilize new unfolding intermediates, showing that Hsp70/40 can directly interact with the folded core of the protein when working as an unfoldase. Our results support an unfolding mechanism where Hsp70 can directly bind to folded protein structures and unfold them upon ATP hydrolysis. These results provide important insights into the regulation of GR by Hsp70/40.

Nanomechanics of the molecular complex between staphylococcal adhesin SpsD and elastin.

Staphylococcus pseudintermedius surface protein SpsD binds to extracellular matrix proteins to invade canine epithelial cells. Using single-molecule experiments, we show that SpsD engages in two modes of interaction with elastin that are tightly controlled by physical stress. Binding is weak (∼100 pN) at low tensile force (i.e. loading rate), but is dramatically enhanced (up to ∼1500 pN) by mechanical tension. Consistent with a "dock, lock, and latch" (DLL) mechanism, this force represents among the highest mechanical strengths known for a non-covalent biological interaction. The transition from weak to strong binding correlates with an increase in molecular stiffness but, surprisingly, with a decrease in molecular extension. This unanticipated mechanical behavior indicates that the adhesin is engaged in two distinct interaction mechanisms. Our results emphasize the crucial role of protein nanomechanics in the adhesion of staphylococci, and illustrate their wide diversity of force-dependent ligand-binding activities. These single-molecule mechanical experiments may contribute to the development of antiadhesion approaches to treat infections caused by S. pseudintermedius and other bacterial pathogens engaged in DLL interactions.

An Active, Ligand-Responsive Pulling Geometry Reports on Internal Signaling between Subdomains of the DnaK Nucleotide-Binding Domain in Single-Molecule Mechanical Experiments.

Single-molecule mechanical experiments have proven to be ideal tools for probing the energetics and mechanics of large proteins and domains. In this paper, we investigate the nucleotide-dependent unfolding mechanics of the nucleotide-binding domain (NBD) of the Hsp70 chaperone DnaK. The NBD binds ADP or ATP in the binding cleft formed by lobe I and lobe II, which consists of two subdomains each. When force is applied to the termini of the NBD, the observed unfolding forces are independent of the nucleotide state. In contrast, when force is applied across the nucleotide-binding pocket, the unfolding forces report specifically on the nucleotide-phosphate state. In this active, ligand-responsive pulling geometry, we observed a bifurcation of the unfolding pathway; the pathway proceeds either through a cooperative "coupled pathway" or through a noncooperative "uncoupled pathway". The partitioning between individual unfolding pathways can be effectively tuned by mutation or by the nucleotide exchange factor GrpE, i.e., by the factors affecting the strength of the lobe I-lobe II interactions within the native NBD. These experiments provide important insight into the molecular origin of the internal signaling between the subdomains of the nucleotide-binding domain of Hsp70 proteins and how signals are efficiently transferred inside the protein molecule.

Anticooperative Binding Governs the Mechanics of Ethidium-Complexed DNA

DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.

Single-molecule dissection of stacking forces in DNA.

We directly measured at the single-molecule level the forces and lifetimes of DNA base-pair stacking interactions for all stack sequence combinations. Our experimental approach combined dual-beam optical tweezers with DNA origami components to allow positioning of blunt-end DNA helices so that the weak stacking force could be isolated. Base-pair stack arrays that lacked a covalent backbone connection spontaneously dissociated at average rates ranging from 0.02 to 500 per second, depending on the sequence combination and stack array size. Forces in the range from 2 to 8 piconewtons that act along the helical direction only mildly accelerated the stochastic unstacking process. The free-energy increments per stack that we estimate from the measured forward and backward kinetic rates ranged from -0.8 to -3.4 kilocalories per mole, depending on the sequence combination. Our data contributes to understanding the mechanics of DNA processing in biology, and it is helpful for designing the kinetics of DNA-based nanoscale devices according to user specifications.

Force-dependent isomerization kinetics of a highly conserved proline switch modulates the mechanosensing region of filamin

Proline switches, controlled by cis-trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. In this study, we use single-molecule mechanical measurements to develop a full kinetic and energetic description of a highly conserved proline switch in the force-sensing domain 20 of human filamin and how prolyl isomerization modulates the force-sensing mechanism. Proline isomerization toggles domain 20 between two conformations. A stable cis conformation with slow unfolding, favoring the autoinhibited closed conformation of filamin's force-sensing domain pair 20-21, and a less stable, uninhibited conformation promoted by the trans form. The data provide detailed insight into the folding mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency in modulating force-sensing, thus combining two previously unconnected regulatory mechanisms, proline switches and mechanosensing.

Rigid DNA Beams for High‐Resolution Single‐Molecule Mechanics

Bridging the gap: Rigid DNA linkers (blue, see picture) between microspheres (green) for high-resolution single-molecule mechanical experiments were constructed using DNA origami. The resulting DNA helical bundles greatly reduce the noise generated in studies of conformation changes using optical tweezers and were applied to study small DNA secondary structures.

Spontaneous Dimerization of Titin Protein Z1Z2 Domains Induces Strong Nanomechanical Anchoring

Muscle elasticity strongly relies on the mechanical anchoring of the giant protein titin to both the sarcomere M-band and the Z-disk. Such strong attachment ensures the reversible dynamics of the stretching-relaxing cycles determining the muscle passive elasticity. Similarly, the design of biomaterials with enhanced elastic function requires experimental strategies able to secure the constituent molecules to avoid mechanical failure. Here we show that an engineered titin-mimicking protein is able to spontaneously dimerize in solution. Our observations reveal that the titin Z1Z2 domains are key to induce dimerization over a long-range distance in proteins that would otherwise remain in their monomeric form. Using single molecule force spectroscopy, we measure the threshold force that triggers the noncovalent transition from protein dimer to monomer, occurring at ∼700 piconewtons. Such extremely high mechanical stability is likely to be a natural protective mechanism that guarantees muscle integrity. We propose a simple molecular model to understand the force-induced dimer-to-monomer transition based on the geometric distribution of forces occurring within a dimeric protein under mechanical tension.

RNA Unwinding by NS3 Helicase: A Statistical Approach

The study of double-stranded RNA unwinding by helicases is a problem of basic scientific interest. One such example is provided by studies on the hepatitis C virus (HCV) NS3 helicase using single molecule mechanical experiments. HCV currently infects nearly 3% of the world population and NS3 is a protein essential for viral genome replication. The objective of this study is to model the RNA unwinding mechanism based on previously published data and study its characteristics and their dependence on force, ATP and NS3 protein concentration. In this work, RNA unwinding by NS3 helicase is hypothesized to occur in a series of discrete steps and the steps themselves occurring in accordance with an underlying point process. A point process driven change point model is employed to model the RNA unwinding mechanism. The results are in large agreement with findings in previous studies. A gamma distribution based renewal process was found to model well the point process that drives the unwinding mechanism. The analysis suggests that the periods of constant extension observed during NS3 activity can indeed be classified into pauses and subpauses and that each depend on the ATP concentration. The step size is independent of external factors and seems to have a median value of 11.37 base pairs. The steps themselves are composed of a number of substeps with an average of about 4 substeps per step and an average substep size of about 3.7 base pairs. An interesting finding pertains to the stepping velocity. Our analysis indicates that stepping velocity may be of two kinds- a low and a high velocity.

The titin-telethonin complex is a directed, superstable molecular bond in the muscle Z-disk

Mechanical stability of bonds and protein interactions has recently become accessible through single molecule mechanical experiments. So far, mechanical information about molecular bond mechanics has been largely limited to a single direction of force application. However, mechanical force acts as a vector in space and hence mechanical stability should depend on the direction of force application. In skeletal muscle, the giant protein titin is anchored in the Z-disk by telethonin. Much of the structural integrity of the Z-disk hinges upon the titin-telethonin bond. In this paper we show that the complex between the muscle proteins titin and telethonin forms a highly directed molecular bond. It is designed to resist ultra-high forces if they are applied in the direction along which it is loaded under physiological conditions, while it breaks easily along other directions. Highly directed molecular bonds match in an ideal way the requirements of tissues subject to mechanical stress.

Can the passive elasticity of muscle be explained directly from the mechanics of individual titin molecules?

Recent progress in understanding the role of titin/connectin in muscle elasticity has been heavily based on results from single molecule mechanical experiments. The shape of force-extension curves from such data is similar to curves from muscle fibres and it has been tempting to assume that muscle elasticity can be extrapolated directly from the single molecule data. In this paper we discuss some of the factors that act on titin in the sarcomere that are likely to preclude such a direct extrapolation.

A model of stereocilia adaptation based on single molecule mechanical studies of myosin I

We have used an optical tweezers-based apparatus to perform single molecule mechanical experiments using the unconventional myosins, Myo1b and Myo1c. The single-headed nature and slow ATPase kinetics of these myosins make them ideal for detailed studies of the molecular mechanism of force generation by acto-myosin. Myo1c exhibits several features that have not been seen using fast skeletal muscle myosin II. (i) The working stroke occurs in two, distinct phases, producing an initial 3 nm and then a further 1.5 nm of movement. (ii) Two types of binding interaction were observed: short-lived ATP-independent binding events that produced no movement and longer-lived, ATP-dependent events that produced a full working stroke. The stiffness of both types of interaction was similar. (iii) In a new type of experiment, using feedback to apply controlled displacements to a single acto-myosin cross-bridge, we found abrupt changes in force during attachment of the acto-Myo1b cross-bridge, a result that is consistent with the classical 'T2' behaviour of single muscle fibres. Given that these myosins might exhibit the classical T2 behaviour, we propose a new model to explain the slow phase of sensory adaptation of the hair cells of the inner ear.

Exploring the energy landscape of GFP by single-molecule mechanical experiments.

We use single-molecule force spectroscopy to drive single GFP molecules from the native state through their complex energy landscape into the completely unfolded state. Unlike many smaller proteins, mechanical GFP unfolding proceeds by means of two subsequent intermediate states. The transition from the native state to the first intermediate state occurs near thermal equilibrium at approximately 35 pN and is characterized by detachment of a seven-residue N-terminal alpha-helix from the beta barrel. We measure the equilibrium free energy cost associated with this transition as 22 k(B)T. Detachment of this small alpha-helix completely destabilizes GFP thermodynamically even though the beta-barrel is still intact and can bear load. Mechanical stability of the protein on the millisecond timescale, however, is determined by the activation barrier of unfolding the beta-barrel out of this thermodynamically unstable intermediate state. High bandwidth, time-resolved measurements of the cantilever relaxation phase upon unfolding of the beta-barrel revealed a second metastable mechanical intermediate with one complete beta-strand detached from the barrel. Quantitative analysis of force distributions and lifetimes lead to a detailed picture of the complex mechanical unfolding pathway through a rough energy landscape.