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-micron 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.

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.

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.

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.

A hallmark of tissue ageing is the irreversible oxidative modifications of its constituent 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 loses completely 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 assay where 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.

The melting transition of deoxyribonucleic acid (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 fibre DNA has been measured as a function of temperature, and its widths and integrated intensities have been interpreted using the Peyrard-Bishop-Dauxois (PBD) 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.PACS numbers: 87.14.gk, 87.15.Zg,87.64.Bx Deoxyribonucleic acid (DNA) is a highly dynamic molecule in which the base pairs, which carry the genetic information, fluctuate widely. This can lead to a temporary breaking of a "closed" pair and a local separation of the two strands. Local openings may be activated by heating. At a certain temperature, local openings of the double helix extend over the full molecule, resulting in a complete separation of the two strands. This is called the "melting" of DNA, and may be considered a very rare example of a one-dimensional (1D) structural phase transition 1,2 . DNA melting attracted attention soon after the discovery of the double helix structure 3,4 and was widely studied because it shows some similarity with DNA unwinding in the cell. There is recent renewed interest in biology due to high resolution melting methods 5 .Despite numerous attempts, the understanding of this transition is still a challenge. One difficulty in making progress is the absence of structural information at the transition. This is because experimental studies of DNA melting, made using techniques such as UV absorbance, circular dichroism and calorimetric studies, do not provide spatial information.Diffraction techniques can provide this information. Indeed, the double-helix structure of DNA was solved by modeling the x-ray diffraction patterns from semicrystalline fibre samples, measured by Franklin et al. 6 . The data were analyzed for peak positions which revealed the double helix structure, and also that the molecules could have different configurational structures 6 . Many configurations are known to exist 7 , however the majority of the work to date has been on so-called 'B-form' DNA.A Bragg peak contains more information than simply its position. Analysis of the shape and width can determine a correlation function and its characteristic length. The integrated intensity gives a measure of the quantity of the sample that scatters coherently. These quantities change dramatically close to a phase transition, and scattering techniques have proved to be excellent probes 8 .We have used neutron scattering to measure the temperature dependence of a strong Bragg peak from a fibre sample of B-form DNA. The data have been analyzed to extract the widths and the integrated intensities as the sample was heated through the melting transition. The structural information was compared with calculations using an adapted form of a mesoscopic statistical mecha...

Advancements in single molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigating how domain folding under force can have physiological roles. 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+β 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 polymer elasticity models, 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 toward deciphering the complexity of multidomain proteins operating under force.

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.