Direct observation of an ensemble of stable collapsed states in the mechanical folding of ubiquitin

Statistical theories of protein folding have long predicted plausible mechanisms for reducing the vast conformational space through distinct ensembles of structures. However, these predictions have remained untested by bulk techniques, because the conformational diversity of folding molecules has been experimentally unapproachable. Owing to recent advances in single molecule force-clamp spectroscopy, we are now able to probe the structure and dynamics of the small protein ubiquitin by measuring its length and mechanical stability during each stage of folding. Here, we discover that upon hydrophobic collapse, the protein rapidly selects a subset of minimum energy structures that are mechanically weak and essential precursors of the native fold. From this much reduced ensemble, the native state is acquired through a barrier-limited transition. Our results support the validity of statistical mechanics models in describing the folding of a small protein on biological timescales.     

Solvent molecules bridge the mechanical unfolding transition state of a protein

We demonstrate a combination of single molecule force spectroscopy and solvent substitution that captures the presence of solvent molecules in the transition state structure. We measure the effect of solvent substitution on the rate of unfolding of the I27 titin module, placed under a constant stretching force. From the force dependency of the unfolding rate, we determine Deltax(u), the distance to the transition state. Unfolding the I27 protein in water gives a Deltax(u) = 2.5 A, a distance that compares well to the size of a water molecule. Although the height of the activation energy barrier to unfolding is greatly increased in both glycerol and deuterium oxide solutions, Deltax(u) depends on the size of the solvent molecules. Upon replacement of water by increasing amounts of the larger glycerol molecules, Deltax(u) increases rapidly and plateaus at its maximum value of 4.4 A. In contrast, replacement of water by the similarly sized deuterium oxide does not change the value of Deltax(u). From these results we estimate that six to eight water molecules form part of the unfolding transition state structure of the I27 protein, and that the presence of just one glycerol molecule in the transition state is enough to lengthen Deltax(u). Our results show that solvent composition is important for the mechanical function of proteins. Furthermore, given that solvent composition is actively regulated in vivo, it may represent an important modulatory pathway for the regulation of tissue elasticity and other important functions in cellular mechanics.     

Water's role in the force-induced unfolding of ubiquitin

In atomic force spectroscopic studies of the elastomeric protein ubiquitin, the β-strands 1-5 serve as the force clamp. Simulations show how the rupture force in the force-induced unfolding depends on the kinetics of water molecule insertion into positions where they can eventually form hydrogen bonding bridges with the backbone hydrogen bonds in the force-clamp region. The intrusion of water into this region is slowed down by the hydrophobic shielding effect of carbonaceous groups on the surface residues of β-strands 1-5, which thereby regulates water insertion prior to hydrogen bond breakage. The experiments show that the unfolding of the mechanically stressed protein is nonexponential due to static disorder. Our simulations show that different numbers and/or locations of bridging water molecules give rise to a long-lived distribution of transition states and static disorder. We find that slowing down the translational (not rotational) motions of the water molecules by increasing the mass of their oxygen atoms, which leaves the force field and thereby the equilibrium structure of the solvent unchanged, increases the average rupture force; however, the early stages of the force versus time behavior are very similar for our "normal" and fictitious "heavy" water models. Finally, we construct six mutant systems to regulate the hydrophobic shielding effect of the surface residues in the force-clamp region. The mutations in the two termini of β-sheets 1-5 are found to determine a preference for different unfolding pathways and change mutant's average rupture force.     

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates

Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.     

A transformation for the mechanical fingerprints of complex biomolecular interactions

Biological processes are carried out through molecular conformational transitions, ranging from the structural changes within biomolecules to the formation of macromolecular complexes and the associations between the complexes themselves. These transitions cover a vast range of timescales and are governed by a tangled network of molecular interactions. The resulting hierarchy of interactions, in turn, becomes encoded in the experimentally measurable “mechanical fingerprints” of the biomolecules, their force–extension curves. However, how can we decode these fingerprints so that they reveal the kinetic barriers and the associated timescales of a biological process? Here, we show that this can be accomplished with a simple, model-free transformation that is general enough to be applicable to molecular interactions involving an arbitrarily large number of kinetic barriers. Specifically, the transformation converts the mechanical fingerprints of the system directly into a map of force-dependent rate constants. This map reveals the kinetics of the multitude of rate processes in the system beyond what is typically accessible to direct measurements. With the contributions from individual barriers to the interaction network now “untangled”, the map is straightforward to analyze in terms of the prominent barriers and timescales. Practical implementation of the transformation is illustrated with simulated biomolecular interactions that comprise different patterns of complexity—from a cascade of activation barriers to competing dissociation pathways.Conformational transitions in biological macromolecules—such as the folding of nucleic acids and proteins or the binding of receptors and their ligands—usually serve as the mechanism that brings biomolecules into their working shape and enables their biological function (1). The conformational dynamics of a biomolecule are governed by its energy, which is described by a hypersurface—the energy landscape—in a space of the multitude of atomic coordinates. The energy landscapes of biological macromolecules are rough and hierarchical: the folded and unfolded (or bound and unbound) conformational states are often separated by a mountainous terrain of barriers (2–4). Remarkably, the prominent features of the landscape can be revealed by pulling the molecule apart: these features manifest themselves as nonmonotonic signatures—rips—in the force–extension curves of the molecule (5). Characteristics of the force–extension curves uniquely identify the biomolecule and thus serve as its “mechanical fingerprints” (6), in which the prominent barriers on the energy landscape are encoded. However, how can we decode the mechanical fingerprints to uncover the locations and heights of the barriers and the associated timescales of biomolecular motion (Fig. 1)? This is the central question addressed in the present paper.Open in a separate windowFig. 1.Conformational transitions in biological macromolecules are often governed by complex energy landscapes. (Upper) A sequence of intermediates separate the native (N) and unfolded (U) states on the free-energy profile. (Lower) Conformational transitions can be resolved as rips (indicated by arrows) in the mechanical fingerprints. The challenge of decoding the fingerprints, so that they reveal the rates and rate-limiting barriers, is addressed in the present study.The realm of biomolecular interactions can be accessed in single-molecule force experiments, which apply a stretching force to a biomolecule and monitor the molecule as it samples its conformations. The force-clamp scheme applies a constant force, while conformational transitions are signaled by abrupt changes in the molecular extension over time. This scheme, repeated at several values of force F, yields the force-dependent rate constant for the transition between states i and j. The force-ramp scheme applies a force that is increased (stretching protocol) or decreased (relaxation protocol) with time, while the transitions are signified by abrupt changes in the force–extension curve. This scheme, repeated at several values of the force-loading rate, yields transition forces F_ij and their probability distributions . Although the rates from the force clamp are, in principle, relatively straightforward to interpret in terms of the kinetic barriers, only a narrow range of forces can be probed in this scheme in practice, which limits access to the full force-dependent profiles of these rates, obstructing their analysis. The force ramp, on the other hand, probes a broader range of forces and is easier to implement, but the analysis of the measured force distributions is not straightforward.An analytical framework for the analysis of the outputs from these two pulling schemes has been developed for the simplest case in which the transition involves a single barrier and is irreversible. Unified expressions for the force-dependent rate of rupture and for the distribution of forces at rupture (7) relate these experimentally measurable quantities to the intrinsic (i.e., zero-force) parameters of the free-energy barrier: its location and height , and the associated rate k₀. Furthermore, mapping that converts into has been established (8). The analytical forms of the expression for (7) and of the mapping of onto (8) make them suitable for the analysis of force-ramp experiments when the conformational transition, or a particular step in the transition, can be viewed as diffusive crossing of a single barrier with no, or no influence from, preceding barriers (9–11). However, conformational transitions in complex biomolecules and macromolecular assemblies usually occur via multiple barriers, as is evident from multiple rips in their force–extension curves. In contrast with the sophistication of the resulting mechanical fingerprints (12–15), there is no analytical theory with which to analyze and interpret such rich behavior. The lack of a theory is evidently due to the difficulty of deriving an analytical expression for the force distribution in multiple-barrier systems. As a result, analytical studies of force-induced molecular transitions in such systems usually focus on the effective rate at constant force (16, 17). An expression for the quantity of relevance to force-ramp experiments—the most probable rupture force at pulling speed V—has been attempted empirically (17): the single-barrier rate was replaced by the multiple-barrier rate in the single-barrier version of the expression for . However, such approach is no longer justified (17) in the force range where two or more barriers have comparable effects on the kinetics.Here, we show that force spectroscopy experiments that probe conformational transitions involving multiple barriers cannot, in general, be approached with the existing analytical tool—the theory for an irreversible single-barrier transition—even when transitions over individual barriers are unambiguously resolved in the experiment. At the same time, deriving a multiple-barrier analog of the expression for the transition force distribution, , is not a feasible approach due to the complexity of the kinetics in multiple-barrier systems. Instead, we propose an approach that bypasses the difficulty of deriving an analytical form of the distribution of transition forces for complex landscapes—by transforming these forces into a form that is straightforward to analyze. This approach is illustrated with several examples representative of the different types of complexity encountered in biomolecular interactions.     

Dissecting the mechanical unfolding of ubiquitin

The unfolding behavior of ubiquitin under the influence of a stretching force recently was investigated experimentally by single-molecule constant-force methods. Many observed unfolding traces had a simple two-state character, whereas others showed clear evidence of intermediate states. Here, we use Monte Carlo simulations to investigate the force-induced unfolding of ubiquitin at the atomic level. In agreement with experimental data, we find that the unfolding process can occur either in a single step or through intermediate states. In addition to this randomness, we find that many quantities, such as the frequency of occurrence of intermediates, show a clear systematic dependence on the strength of the applied force. Despite this diversity, one common feature can be identified in the simulated unfolding events, which is the order in which the secondary-structure elements break. This order is the same in two- and three-state events and at the different forces studied. The observed order remains to be verified experimentally but appears physically reasonable.     

Trans-bonded pairs of E-cadherin exhibit a remarkable hierarchy of mechanical strengths

Classical cadherins are primary mediators of calcium-dependent cell interactions in multicellular organisms. Organized in five tandemly repeated E-cadherin (EC) modules, the extracellular segments of these membrane-spanning glycoproteins interact homophilically between opposing cells to create highly regulated patterns of attachment stabilized by cytoskeletal elements inside the cells. Despite many structural and functional studies, a significant controversy exists in regard to the organization of cadherin binding in adhesion sites. Supported by considerable evidence, perhaps the most widely held view is that opposing N-terminal EC1-EC2 (EC12) domains form a "zipper" of bonds. However, immobilized on two atomically smooth surfaces and pushed to adhesive contact, opposing cadherins become fully interdigitated and unbind through three discrete jumps comparable with domain dimensions when pulled apart. So the question remains as to whether mechanical adhesion strength emanates solely from interactions between the peripheral N-terminal domains or involves multiple overlapping domains. It is also unclear whether a primary adhesion complex is formed by a single opposing pair of cadherins or whether the complex involves a more complicated network of cis-bonded multimers. To address these questions, we used a special jump/ramp mode of force spectroscopy to test isolated pairwise interactions between recombinant fragments of ECs. Besides the formation of strong trans-bonded dimers, we find a remarkable hierarchy of rupture strengths for bonds between the full five-domain fragments that suggests multiple mechanical functions for cadherins, perhaps providing distinct properties needed for transient-specific recognition as well as stable tissue formation.     

Early hydrogen-bonding events in the folding reaction of ubiquitin.

The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogen-deuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 back-bone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the beta-sheet and the alpha-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slow-folding species are less prominent.     

How the hydrophobic factor drives protein folding

How hydrophobicity (HY) drives protein folding is studied. The 1971 Nozaki–Tanford method of measuring HY is modified to use gases as solutes, not crystals, and this makes the method easy to use. Alkanes are found to be much more hydrophobic than rare gases, and the two different kinds of HY are termed intrinsic (rare gases) and extrinsic (alkanes). The HY values of rare gases are proportional to solvent-accessible surface area (ASA), whereas the HY values of alkanes depend on special hydration shells. Earlier work showed that hydration shells produce the hydration energetics of alkanes. Evidence is given here that the transfer energetics of alkanes to cyclohexane [Wolfenden R, Lewis CA, Jr, Yuan Y, Carter CW, Jr (2015) Proc Natl Acad Sci USA 112(24):7484–7488] measure the release of these shells. Alkane shells are stabilized importantly by van der Waals interactions between alkane carbon and water oxygen atoms. Thus, rare gases cannot form this type of shell. The very short (approximately picoseconds) lifetime of the van der Waals interaction probably explains why NMR efforts to detect alkane hydration shells have failed. The close similarity between the sizes of the opposing energetics for forming or releasing alkane shells confirms the presence of these shells on alkanes and supports Kauzmann''s 1959 mechanism of protein folding. A space-filling model is given for the hydration shells on linear alkanes. The model reproduces the n values of Jorgensen et al. [Jorgensen WL, Gao J, Ravimohan C (1985) J Phys Chem 89:3470–3473] for the number of waters in alkane hydration shells.When Kauzmann published his classic 1959 paper (1) on a new hydrophobic factor that drives protein folding, he gave examples from the literature (his table 3) of the energetics of transferring alkanes and aromatic hydrocarbons between various organic solvents and water. These examples show that the transfer free energy is favorable and sizable when a hydrocarbon solute is transferred from water to an organic solvent. Finding a favorable transfer free energy from water to an organic solvent prompted Kauzmann (1, 2) to suggest a protein-folding mechanism in which hydrocarbon side chains of the unfolded protein are driven to leave water and enter the folded protein interior because the interior is water free.Tanford (3) in 1962 then showed how the hydrophobic factor can be evaluated quantitatively [as the hydrophobicity (HY)] for amino acid side chains by using their solubilities in ethanol and water. Tanford (3) showed that the transfer free energy from water to ethanol can be found from the solubilities of the solute in water and ethanol, and he pointed out that the required solubility data are given in the book by Cohn and Edsall (4). By making free-energy calculations from these data, Tanford (3) in 1962 began the quantitative study of HY, and he argued that it is the key to understanding how proteins fold. In 1971, Nozaki and Tanford (5) gave the name HY to this type of analysis. The long-standing meaning of the term hydrophobic (water-hating) is that a hydrophobic solute is much more soluble in most organic solvents than in water (6). Nozaki and Tanford (5) used this meaning to develop a method of measuring HY values from the solute''s solubilities in water and ethanol, or other reference solvent. In 1971, they gave their measurements of HY values (5) for 12 amino acid side chains and for the peptide unit. Their 1971 HY values agree fairly well with the ones Tanford (3) gave in 1962, based on literature values for the solubilities.The 1971 Nozaki–Tanford paper (5) became an instant classic, and their method of measuring HY values was widely accepted. However, the 1971 Nozaki–Tanford method is difficult to use and was not used after 1971, not even by Tanford. To make the Nozaki–Tanford method of measuring HY values simpler to use, we modified the method to use gases rather than crystalline side chains as solutes. Some of the crystalline amino acids studied in 1971 by Nozaki and Tanford (5) were insoluble in both reference solvents, ethanol and dioxane, used by them. Thus, they had to make solubility measurements in mixtures of water and ethanol (or dioxane) to obtain measurable solubilities, and they needed to make difficult extrapolations to obtain solubility results for 100% ethanol or 100% dioxane. Using the modified method given here, with gases as solutes, there is no similar solubility problem.HY values are measured here for alkanes and rare gases. Alkanes are found to be much more hydrophobic than rare gases; in fact, there are two different kinds of HY. This result was expected from the proposal by Jorgensen et al. (7), who showed that van der Waals (vdW) interactions between alkane carbon and water oxygen atoms tether a fixed number (n) of water molecules to each of the seven alkanes they studied. Moreover, Jorgensen et al. (7) measured the Lennard–Jones potential of the C...O vdW interaction and found that it is quite strong. Jorgensen et al. (7) proposed that these tethered water molecules serve as Kauzmann''s (1, 2) hydration shells, which contribute to the solute''s HY. Thus, alkanes were expected to be more hydrophobic than rare gases because rare gases lack carbon atoms and cannot form these tethered water molecules.     

Differences in the folding transition state of ubiquitin indicated by phi and psi analyses

We compare the folding transition state (TS) of ubiquitin previously identified by using psi analysis to that determined by using analysis. Both methods attempt to identify interactions and their relative populations at the rate-limiting step for folding. The TS ensemble derived from psi analysis has an extensive native-like chain topology, with a four-stranded beta-sheet network and a portion of the major helix. According to analysis, however, the TS is much smaller and more polarized, with only a local helix/hairpin motif. We find that structured regions can have values far from unity, the canonical value for such sites, because of structural relaxation of the TS. Consequently, these sites may be incorrectly interpreted as contributing little to the structure of the TS. These results stress the need for caution when interpreting and drawing conclusions from analysis alone and highlight the need for more specific tools for examining the structure and energetics of the TS ensemble.     

Ionic effects on viral DNA packaging and portal motor function in bacteriophage phi 29

In many viruses, DNA is confined at such high density that its bending rigidity and electrostatic self-repulsion present a strong energy barrier in viral assembly. Therefore, a powerful molecular motor is needed to package the DNA into the viral capsid. Here, we investigate the role of electrostatic repulsion on single DNA packaging dynamics in bacteriophage phi 29 via optical tweezers measurements. We show that ionic screening strongly affects the packing forces, confirming the importance of electrostatic repulsion. Separately, we find that ions affect the motor function. We separate these effects through constant force measurements and velocity versus load measurements at both low and high capsid filling. Regarding motor function, we find that eliminating free Mg(2+) blocks initiation of packaging. In contrast, Na(+) is not required, but it increases the motor velocity by up to 50% at low load. Regarding internal resistance, we find that the internal force was lowest when Mg(2+) was the dominant ion or with the addition of 1 mM Co(3+). Forces resisting DNA confinement were up to approximately 80% higher with Na(+) as the dominant counterion, and only approximately 90% of the genome length could be packaged in this condition. The observed trend of the packing forces is in accord with that predicted by DNA charge-screening theory. However, the forces are up to six times higher than predicted by models that assume coaxial spooling of the DNA and interaction potentials derived from DNA condensation experiments. The forces are also severalfold higher than ejection forces measured with bacteriophage lambda.     

Probing osmolyte participation in the unfolding transition state of a protein

Understanding the molecular mechanisms of osmolyte protection in protein stability has proved to be challenging. In particular, little is known about the role of osmolytes in the structure of the unfolding transition state of a protein, the main determinant of its dynamics. We have developed an experimental protocol to directly probe the transition state of a protein in a range of osmolyte environments. We use an atomic force microscope in force-clamp mode to apply mechanical forces to the protein I27 and obtain force-dependent rate constants of protein unfolding. We measure the distance to the unfolding transition state, Δx(u), along a 1D reaction coordinate imposed by mechanical force. We find that for the small osmolytes, ethylene glycol, propylene glycol, and glycerol, Δx(u) scales with the size of the molecule, whereas for larger osmolytes, sorbitol and sucrose, Δx(u) remains the same as that measured in water. These results are in agreement with steered molecular dynamics simulations that show that small osmolytes act as solvent bridges in the unfolding transition state structure, whereas only water molecules act as solvent bridges in large osmolyte environments. These results demonstrate that novel force protocols combined with solvent substitution can directly probe angstrom changes in unfolding transition state structure. This approach creates new opportunities to gain molecular level understanding of the action of osmolytes in biomolecular processes.     

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10° accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90° for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.     

Weak temporal signals can synchronize and accelerate the transition dynamics of biopolymers under tension

In addition to thermal noise, which is essential to promote conformational transitions in biopolymers, the cellular environment is replete with a spectrum of athermal fluctuations that are produced from a plethora of active processes. To understand the effect of athermal noise on biological processes, we studied how a small oscillatory force affects the thermally induced folding and unfolding transition of an RNA hairpin, whose response to constant tension had been investigated extensively in both theory and experiments. Strikingly, our molecular simulations performed under overdamped condition show that even at a high (low) tension that renders the hairpin (un)folding improbable, a weak external oscillatory force at a certain frequency can synchronously enhance the transition dynamics of RNA hairpin and increase the mean transition rate. Furthermore, the RNA dynamics can still discriminate a signal with resonance frequency even when the signal is mixed among other signals with nonresonant frequencies. In fact, our computational demonstration of thermally induced resonance in RNA hairpin dynamics is a direct realization of the phenomena called stochastic resonance and resonant activation. Our study, amenable to experimental tests using optical tweezers, is of great significance to the folding of biopolymers in vivo that are subject to the broad spectrum of cellular noises.     

The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

Gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. Supercoiling is catalyzed via a strand-passage mechanism, in which the gate DNA (gDNA) is transiently cleaved, and a second DNA segment, the transfer DNA (tDNA), is passed through the gap before the gDNA is religated. Strand passage requires an opening of the so-called DNA-gate by ≈2 nm. A single-molecule FRET study reported equal populations of open and closed DNA-gate in topoisomerase II. We present here single-molecule FRET experiments that monitor the conformation of DNA bound to the DNA-gate of Bacillus subtilis gyrase and the conformation of the DNA-gate itself. DNA bound to gyrase adopts two different conformations, one slightly, one severely distorted. DNA distortion requires cleavage, but neither ATP nor the presence of a tDNA. At the same time, the DNA-gate of gyrase is predominantly in the closed conformation. In agreement with the single molecule data and with the danger of dsDNA breaks for genome integrity, <5% of cleavage complexes are detected in equilibrium. Quinolone inhibitors favor DNA cleavage by B. subtilis gyrase, but disfavor DNA distortion, and the DNA-gate remains in the closed conformation. Our results demonstrate that DNA binding, distortion and cleavage, and gate-opening are mechanistically distinct events. During the relaxation and supercoiling reactions, gyrase with an open DNA-gate is not significantly populated, consistent with gate-opening as a very rare event that only occurs briefly to allow for strand passage.     

Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational-active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca(2+) resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.