On the distance to the transition state of protein folding in optical tweezers experiments.

The distance to the transition state ( ) is an important parameter for understanding the energy landscape of chemical reactions. In protein folding, represents the distance to the high energy structure between folded and unfolded states. This correlates with the deformation of the protein as it crosses the energy barrier defining its rigidity.

Mechanical force activates the light-dependent channels TRP and TRPL in excised patches from the rhabdomere of Drosophila photoreceptors.

Drosophila phototransduction in light-sensitive microvilli involves a metabotropic signaling cascade. Photoisomerized rhodopsin couples to G-protein, activating phospholipase C, which cleaves phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate, diacylglycerol (DAG) and a proton. DAG is converted into phosphatidic acid by DAG-kinase and metabolized to L-linoleoyl glycerol (2-LG) by DAG-lipase.

Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers.

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction.

Towards the understanding of molecular motors and its relationship with local unfolding.

Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis.

Mutations in yeast Pcf11, a conserved protein essential for mRNA 3' end processing and transcription termination, elicit the Environmental Stress Response.

The Pcf11 protein is an essential subunit of the large complex that cleaves and polyadenylates eukaryotic mRNA precursor. It has also been functionally linked to gene-looping, termination of RNA Polymerase II (Pol II) transcripts, and mRNA export. We have examined a poorly characterized but conserved domain (amino acids 142-225) of the Saccharomyces cerevisiae  Pcf11 and found that while it is not needed for mRNA 3' end processing or termination downstream of the poly(A) sites of protein-coding genes, its presence improves the interaction with Pol II and the use of transcription terminators near gene promoters.

Bioinformatics, thermodynamics, and mechanical resistance of the FtsZ-ZipA complex of Escherichia coli supports a highly dynamic protein interaction in the divisome.

In most microorganisms, cell division is guided by the divisome, a multiprotein complex that assembles at the equator of the cell and is responsible for the synthesis of new cell wall material. FtsZ, the first protein to assemble into this complex forms protofilaments in the cytosol which are anchored to the inner side of the cytosolic membrane by the proteins ZipA and FtsA. FtsZ protofilaments generate a force that deforms the cytosolic membrane and may contribute to the constriction force that leads to the septation of the cell.

Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate.

BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding.

Temperature dependent mechanical unfolding and refolding of a protein studied by thermo-regulated optical tweezers.

Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic contributions are obtained by quantifying the conformational equilibrium based on melting experiments performed in bulk conditions. Such experiments are suitable only for those small single-domain proteins whose side reactions of irreversible aggregation are unlikely to occur.

Determination of protein-protein interactions at the single-molecule level using optical tweezers.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule () experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based () methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems.

Identification of VEGFR2 as the Histatin-1 receptor in endothelial cells.

Histatin-1 is a salivary peptide with antimicrobial and wound healing promoting activities, which was previously shown to stimulate angiogenesis in vitro and in vivo via inducing endothelial cell migration. The mechanisms underlying the proangiogenic effects of Histatin-1 remain poorly understood and specifically, the endothelial receptor for this peptide, is unknown. Based on the similarities between Histatin-1-dependent responses and those induced by the prototypical angiogenic receptor, vascular endothelial growth factor receptor 2 (VEGFR2), we hypothesized that VEGFR2 is the Histatin-1 receptor in endothelial cells.

Standardized and reproducible measurement of decision-making in mice.

Progress in science requires standardized assays whose results can be readily shared, compared, and reproduced across laboratories. Reproducibility, however, has been a concern in neuroscience, particularly for measurements of mouse behavior. Here, we show that a standardized task to probe decision-making in mice produces reproducible results across multiple laboratories.

Application of Force to a Syndecan-4 Containing Complex With Thy-1-αβ Integrin Accelerates Neurite Retraction.

Inflammation contributes to the genesis and progression of chronic diseases, such as cancer and neurodegeneration. Upregulation of integrins in astrocytes during inflammation induces neurite retraction by binding to the neuronal protein Thy-1, also known as CD90. Additionally, Thy-1 alters astrocyte contractility and movement by binding to the mechano-sensors αβ integrin and Syndecan-4.

PKD2/polycystin-2 induces autophagy by forming a complex with BECN1.

Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD).

Structural characterization of the saxitoxin-targeting APTSTX1 aptamer using optical tweezers and molecular dynamics simulations.

Optical tweezers have enabled the exploration of picoNewton forces and dynamics in single-molecule systems such as DNA and molecular motors. In this work, we used optical tweezers to study the folding/unfolding dynamics of the APTSTX1-aptamer, a single-stranded DNA molecule with high affinity for saxitoxin (STX), a lethal neurotoxin. By measuring the transition force during (un)folding processes, we were able to characterize and distinguish the conformational changes of this aptamer in the presence of magnesium ions and toxin.

Mechanical properties of BiP protein determined by nano-rheology.

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD.

Identifying Chaotropic and Kosmotropic Agents by Nanorheology.

Chemical agents are classified into chaotropes (disorder inducing) and kosmotropes (order inducing) based on their ability to destabilize or stabilize, respectively, the structure of hydrated macromolecules and their solutions. Here, we examine the effect of such agents on the mechanical stiffness of the hydration layer of proteins, measured by nanorheology. We examine four different agents and conclude that chaotropic substances induce the overall softening of the protein-hydration layer system, whereas the kosmotropic substances induce stiffening.

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure.

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.

Author Correction: The energy cost of polypeptide knot formation and its folding consequences.

The original version of this article contained an error in the spelling of the author Christian A.M. Wilson, which was incorrectly given as Christian M.

Single-molecule measurements of the effect of force on Thy-1/αvβ3-integrin interaction using nonpurified proteins.

Thy-1 and αvβ3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvβ3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell-cell adhesion. Nonetheless, how the Thy-1/αvβ3 interactions respond to mechanical cues is an unresolved issue.

The energy cost of polypeptide knot formation and its folding consequences.

Knots are natural topologies of chains. Yet, little is known about spontaneous knot formation in a polypeptide chain-an event that can potentially impair its folding-and about the effect of a knot on the stability and folding kinetics of a protein. Here we used optical tweezers to show that the free energy cost to form a trefoil knot in the denatured state of a polypeptide chain of 120 residues is 5.

Single molecule force spectroscopy reveals the effect of BiP chaperone on protein folding.

BiP (Immunoglobulin Binding Protein) is a member of the Hsp70 chaperones that participates in protein folding in the endoplasmic reticulum. The function of BiP relies on cycles of ATP hydrolysis driving the binding and release of its substrate proteins. It still remains unknown how BiP affects the protein folding pathway and there has been no direct demonstration showing which folding state of the substrate protein is bound by BiP, as previous work has used only peptides.

Novel indolizino[8,7-b]indole hybrids as anti-small cell lung cancer agents: Regioselective modulation of topoisomerase II inhibitory and DNA crosslinking activities.

A novel series of bis(hydroxymethyl)indolizino[8,7-b]indole hybrids composed of β-carboline (topoisomerase I/II inhibition) and bis(hydroxymethyl)pyrrole (DNA cross-linking) are synthesized for antitumor evaluation. Of tumor cell lines tested, small cell lung cancer (SCLC) cell lines are the most sensitive to the newly synthesized compounds. These hybrids induce cell cycle arrest at the G2/M phase, trigger tumor cell apoptotic death, and display diverse mechanisms of action involving topoisomerase II (Topo II) inhibition and induction of DNA cross-linking.

New visible and selective DNA staining method in gels with tetrazolium salts.

DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate.