Detecting protein folding by thermal fluctuations of microcantilevers.

The accurate characterization of proteins in both their native and denatured states is essential to effectively understand protein function, folding and stability. As a proof of concept, a micro rheological method is applied, based on the characterization of thermal fluctuations of a micro cantilever immersed in a bovine serum albumin solution, to assess changes in the viscosity associated with modifications in the protein's structure under the denaturant effect of urea. Through modeling the power spectrum density of the cantilever's fluctuations over a broad frequency band, it is possible to implement a fitting procedure to accurately determine the viscosity of the fluid, even at low volumes. Increases in viscosity during the denaturant process are identified using the assumption that the protein is a hard sphere, with a hydrodynamic radius that increases during unfolding. This is modeled accordingly through the Einstein-Batchelor formula. The Einstein-Batchelor formula estimates are verified through dynamic light scattering, which measures the hydrodynamic radius of proteins. Thus, this methodology is proven to be suitable for the study of protein folding in samples of small size at vanishing shear stresses.

Rosmarinic acid prevents fibrillization and diminishes vibrational modes associated to ß sheet in tau protein linked to Alzheimer's disease.

Alzheimer's disease is a common tauopathy where fibril formation and aggregates are the hallmark of the disease. Efforts targeting amyloid-ß plaques have succeeded to remove plaques but failed in clinical trials to improve cognition; thus, the current therapeutic strategy is at preventing tau aggregation. Here, we demonstrated that four phenolic diterpenoids and rosmarinic acid inhibit fibrillization. Since, rosmarinic acid was the most active compound, we observe morphological changes in atomic force microscopy images after treatment. Hence, rosmarinic acid leads to a decrease in amide regions I and III, indicating that rosmarinic acid prevents ß-sheet assembly. Molecular docking study inside the steric zipper model of the hexapeptide 306VQIVYK311 involved in fibrillization and ß sheet formation, suggests that rosmarinic acid binds to the steric zipper with similar chemical interactions with respect to those observed for orange G, a known pharmacofore for amyloid.

Low thermal fluctuations in a system heated out of equilibrium.

We study the mechanical fluctuations of a micrometer sized silicon cantilever subjected to a strong heat flow, thus having a highly nonuniform local temperature. In this nonequilibrium steady state, we show that fluctuations are equivalent to the thermal noise of a cantilever at equilibrium around room temperature, while its mean local temperature is several hundred degrees higher. Changing the mechanical dissipation by adding a coating to the cantilever, we recover the expected rise of fluctuations with the mean temperature. Our work demonstrates that inhomogeneous dissipation mechanisms can decouple the amplitude of thermal fluctuations from the average temperature. This property could be useful to understand out-of-equilibrium fluctuating systems, or to engineer low noise instruments.

Pulling on super paramagnetic beads with micro cantilevers: single molecule mechanical assay application.

This paper demonstrates that it is possible to trap and release a super paramagnetic micro bead by fixing three super paramagnetic micro beads in a triangular array at the sensitive end of a micro cantilever, and by simply switching on/off an external magnetic field. To provide evidence of this principle we trap a micro bead that is attached to the free end of single DNA molecule and that has been previously fixed at the other end to a glass surface, using the standard sample preparation protocol of magnetic tweezers assays. The switching process is reversible which preserves the integrity of the tethered molecule, and a local force applied over the tethered bead excludes the neighbouring beads from the magnetic trap. We have developed a quadrature phase interferometer which is able to perform under fluid environments to accurately measure small deflections, which permits the exploration of DNA elasticity. Our results agree with measurements from magnetic tweezer assays performed under similar conditions. Furthermore, compared to the magnetic tweezer methodology, the combination of the magnetic trap with a suitable measurement system for cantilever deflection, allows for the exploration of a wide range of forces using a local method that has an improved temporal resolution.

Quadrature phase interferometer for high resolution force spectroscopy.

In this article, we present a deflection measurement setup for Atomic Force Microscopy (AFM). It is based on a quadrature phase differential interferometer: we measure the optical path difference between a laser beam reflecting above the cantilever tip and a reference beam reflecting on the static base of the sensor. A design with very low environmental susceptibility and another allowing calibrated measurements on a wide spectral range are described. Both enable a very high resolution (down to 2.5×10(-15) m/√Hz), illustrated by thermal noise measurements on AFM cantilevers. They present an excellent long-term stability and a constant sensitivity independent of the optical phase of the interferometer. A quick review shows that our precision is equaling or out-performing the best results reported in the literature, but for a much larger deflection range, up to a few µm.