Observing G4 formation and its resolution by Pif1 in real time by manipulation under magnetic tweezers.

G-quadruplexes (G4s) are nucleic acids secondary structures that may form in guanine-rich sequences, either intra or inter-molecularly. Ability of a primary sequence to form a G4 can be predicted computationally with an improving accuracy as well as tested in bulk using biophysical measurements. As a result, G4 density maps have been devised for a large number of genomes from all life kingdoms. Experimental validation of the formation of G4s in vivo however remains indirect and relies on their stabilization with small molecules, antibodies or proteins, or mutational studies, in order to measure downstream effects on gene expression or genome stability for example. Although numerous techniques exist to observe spontaneous formation of G4s in single-stranded DNA, observing G4 formation in double-stranded DNA (dsDNA) is more challenging. However, it is particularly relevant to understand if a given G4 sequence forms stably in a dsDNA context, if it is stable enough to dock proteins or pose a challenge to molecular motors such as helicases or polymerases. In essence, G4s can be a threat to genomic stability but carry as well as the potential to be elements of a structural language in the non-replicating genome. To study quantitatively the formation dynamics and stability of single intramolecular G4s embedded in dsDNA, we have adapted techniques of DNA manipulation under magnetic tweezers. This technique also allows to study encounters of molecular motors with G4 at a single molecule resolution, in order to gain insight into the specificity of G4 resolution by molecular motors, and its efficiency. The procedures described here include the design of the G4 substrate, the study of G4 formation probability and lifetime in dsDNA, as well as procedures to characterize the encounter between the Pif1 helicase and a G4 until G4 resolution. The procedures that we described here can easily be extended to the study of other G4s or molecular motors.

Strand switching mechanism of Pif1 helicase induced by its collision with a G-quadruplex embedded in dsDNA.

G-rich sequences found at multiple sites throughout all genomes may form secondary structures called G-quadruplexes (G4), which act as roadblocks for molecular motors. Among the enzymes thought to process these structures, the Pif1 DNA helicase is considered as an archetypical G4-resolvase and its absence has been linked to G4-related genomic instabilities in yeast. Here we developed a single-molecule assay to observe Pif1 opening a DNA duplex and resolving the G4 in real time. In support of former enzymological studies, we show that the helicase reduces the lifetime of G4 from hours to seconds. However, we observe that in the presence of a G4, Pif1 exhibits a strong strand switching behavior, which can lead to Pif1 escaping G4 resolution, depending on the structural context surrounding the substrate. This behavior is also detected in the presence of other roadblocks (LNA or RNA). We propose that the efficiency of Pif1 to remove a roadblock (G4 or other) is affected by its strand switching behavior and depends on the context surrounding the obstacle. We discuss how this switching behavior may explain several aspects of Pif1 substrate preference and affect its activity as a G4 resolvase in vivo.

Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding.

Fluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein's natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

Segmentation and the Entropic Elasticity of Modular Proteins.

Single-molecule force spectroscopy utilizes polyproteins, which are composed of tandem modular domains, to study their mechanical and structural properties. Under the application of external load, the polyproteins respond by unfolding and refolding domains to acquire the most favored extensibility. However, unlike single-domain proteins, the sequential unfolding of the each domain modifies the free energy landscape (FEL) of the polyprotein nonlinearly. Here we use force-clamp (FC) spectroscopy to measure unfolding and collapse-refolding dynamics of polyubiquitin and poly(I91). Their reconstructed unfolding FEL involves hundreds of kB T in accumulating work performed against conformational entropy, which dwarfs the ∼30 kB T that is typically required to overcome the free energy difference of unfolding. We speculate that the additional entropic energy caused by segmentation of the polyprotein to individual proteins plays a crucial role in defining the "shock absorber" properties of elastic proteins such as the giant muscle protein titin.

Trigger factor chaperone acts as a mechanical foldase.

Proteins fold under mechanical forces in a number of biological processes, ranging from muscle contraction to co-translational folding. As force hinders the folding transition, chaperones must play a role in this scenario, although their influence on protein folding under force has not been directly monitored yet. Here, we introduce single-molecule magnetic tweezers to study the folding dynamics of protein L in presence of the prototypical molecular chaperone trigger factor over the range of physiological forces (4-10 pN). Our results show that trigger factor increases prominently the probability of folding against force and accelerates the refolding kinetics. Moreover, we find that trigger factor catalyzes the folding reaction in a force-dependent manner; as the force increases, higher concentrations of trigger factor are needed to rescue folding. We propose that chaperones such as trigger factor can work as foldases under force, a mechanism which could be of relevance for several physiological processes.Proteins fold under mechanical force during co-translational folding at the ribosome. Here, the authors use single molecule magnetic tweezers to study the influence of chaperones on protein folding and show that the ribosomal chaperone trigger factor acts as a mechanical foldase by promoting protein folding under force.

Proteins Breaking Bad: A Free Energy Perspective.

Protein aging may manifest as a mechanical disease that compromises tissue elasticity. As proved recently, while proteins respond to changes in force with an instantaneous elastic recoil followed by a folding contraction, aged proteins break bad, becoming unstructured polymers. Here, we explain this phenomenon in the context of a free energy model, predicting the changes in the folding landscape of proteins upon oxidative aging. Our findings validate that protein folding under force is constituted by two separable components, polymer properties and hydrophobic collapse, and demonstrate that the latter becomes irreversibly blocked by oxidative damage. We run Brownian dynamics simulations on the landscape of protein L octamer, reproducing all experimental observables, for a naive and damaged polyprotein. This work provides a unique tool to understand the evolving free energy landscape of elastic proteins upon physiological changes, opening new perspectives to predict age-related diseases in tissues.

Mechanical Deformation Accelerates Protein Ageing.

A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week-long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.

Multidomain proteins under force.

Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + ß fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics.

Under physiological conditions, protein oxidation and misfolding occur with very low probability and on long times scales. Single-molecule techniques provide the ability to distinguish between properly folded and damaged proteins that are otherwise masked in ensemble measurements. However, at physiological conditions these rare events occur with a time constant of several hours, inaccessible to current single-molecule approaches. Here we present a magnetic-tweezers-based technique that allows, for the first time, the study of folding of single proteins during week-long experiments. This technique combines HaloTag anchoring, sub-micrometer positioning of magnets, and an active correction of the focal drift. Using this technique and protein L as a molecular template, we generate a magnet law by correlating the distance between the magnet and the measuring paramagnetic bead with unfolding/folding steps. We demonstrate that, using this magnet law, we can accurately measure the dynamics of proteins over a wide range of forces, with minimal dispersion from bead to bead. We also show that the force calibration remains invariant over week-long experiments applied to the same single proteins. The approach demonstrated in this Article opens new, exciting ways to examine proteins on the "human" time scale and establishes magnetic tweezers as a valuable technique to study low-probability events that occur during protein folding under force.

Melting of highly oriented fiber DNA subjected to osmotic pressure.

A pilot study of the possibility to investigate temperature-dependent neutron scattering from fiber-DNA in solution is presented. The study aims to establish the feasibility of experiments to probe the influence of spatial confinement on the structural correlation and the formation of denatured bubbles in DNA during the melting transition. Calorimetry and neutron scattering experiments on fiber samples immersed in solutions of poly(ethylene glycol) (PEG) prove that the melting transition occurs in these samples, that the transition is reversible to some degree, and that the transition is broader in temperature than for humidified fiber samples. The PEG solutions apply an osmotic pressure that maintains the fiber orientation, establishing the feasibility of future scattering experiments to study the melting transition in these samples.

The elastic free energy of a tandem modular protein under force.

Recent studies have provided a theoretical framework for including entropic elasticity in the free energy landscape of proteins under mechanical force. Accounting for entropic elasticity using polymer physics models has helped explain the hopping behavior seen in single molecule experiments in the low force regime. Here, we expand on the construction of the free energy of a single protein domain under force proposed by Berkovich et al. to provide a free energy landscape for N tandem domains along a continuous polypeptide. Calculation of the free energy of individual domains followed by their concatenation provides a continuous free energy landscape whose curvature is dominated by the worm-like chain at forces below 20 pN. We have validated our free energy model using Brownian dynamics and reproduce key features of protein folding. This free energy model can predict the effects of changes in the elastic properties of a multidomain protein as a consequence of biological modifications such as phosphorylation or the formation of disulfide bonds. This work lays the foundations for the modeling of tissue elasticity, which is largely determined by the properties of tandem polyproteins.

Continuity of states between the cholesteric → line hexatic transition and the condensation transition in DNA solutions.

A new method of finely temperature-tuning osmotic pressure allows one to identify the cholesteric → line hexatic transition of oriented or unoriented long-fragment DNA bundles in monovalent salt solutions as first order, with a small but finite volume discontinuity. This transition is similar to the osmotic pressure-induced expanded → condensed DNA transition in polyvalent salt solutions at small enough polyvalent salt concentrations. Therefore there exists a continuity of states between the two. This finding, together with the corresponding empirical equation of state, effectively relates the phase diagram of DNA solutions for monovalent salts to that for polyvalent salts and sheds some light on the complicated interactions between DNA molecules at high densities.

Ionic mobility in DNA films studied by dielectric spectroscopy.

Double-helix DNA molecules can be found under different conformational structures driven by ionic and hydration surroundings. Usually, only the B-form of DNA, which is the only form stable in aqueous solution, can be studied by dielectric measurements. Here, the dielectric responses of DNA molecules in the A- and B-form, oriented co-linearly within fibres assembled in a film have been analyzed. The dielectric dispersion, permittivity and dissipation factor, have been measured as a function of frequency, strength voltage, time, temperature and nature of the counter-ions. Besides a high electrode polarization component, two relaxation peaks have been observed and fitted by two Cole-Cole relaxation terms. In the frequency range that we investigated (0.1 Hz to 5 ·10(6) Hz) the dielectric properties are dominated by the mobility and diffusivity of the counter-ions and their interactions with the DNA molecules, which can therefore be characterized for the A- and B-forms of DNA.

Purification of A-form DNA fiber samples by the removal of B-form DNA residues.

To date, fiber diffraction on A-form NaDNA has always shown a B-form contamination. Here we have used optic microscopy, calorimetry, and neutron scattering techniques to define a method to obtain DNA fibres samples whose molecules are purely in the A-form. When the impure sample is heated to 320 K, the DNA molecules in the B-form undergo a transition into the A-form. Our studies have modified the accepted phase diagram for NaDNA films by including the dependence of temperature crucial for the purification of A-form samples by removal of B-form contamination.

Glassy behavior of denatured DNA films studied by differential scanning calorimetry.

We use differential scanning calorimetry (DSC) to study the properties of DNA films, made of oriented fibers, heated above the thermal denaturation temperature of the double helical form. The films show glassy properties that we investigate in two series of experiments, a slow cooling at different rates followed by a DSC scan upon heating and aging at a temperature below the glass transition. Introducing the fictive temperature to characterize the glass allows us to derive quantitative information on the relaxations of the DNA films, in particular to evaluate their enthalpy barrier. A comparison with similar aging studies on PVAc highlights some specificities of the DNA samples.

Structural correlations and melting of B-DNA fibers.

Despite numerous attempts, understanding the thermal denaturation of DNA is still a challenge due to the lack of structural data on the transition since standard experimental approaches to DNA melting are made in solution and do not provide spatial information. We report a measurement using neutron scattering from oriented DNA fibers to determine the size of the regions that stay in the double-helix conformation as the melting temperature is approached from below. A Bragg peak from the B form of DNA is observed as a function of temperature and its width and integrated intensity are measured. These results, complemented by a differential calorimetry study of the melting of B-DNA fibers as well as electrophoresis and optical observation data, are analyzed in terms of a one-dimensional mesoscopic model of DNA.

Thermal denaturation of DNA studied with neutron scattering.

The melting transition of DNA, whereby the strands of the double-helix structure completely separate at a certain temperature, has been characterized using neutron scattering. A Bragg peak from B-form fiber DNA has been measured as a function of temperature, and its widths and integrated intensities have been interpreted using the Peyrard-Bishop-Dauxois model with only one free parameter. The experiment is unique, as it gives spatial correlation along the molecule through the melting transition where other techniques cannot.