The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding.

One of the most exciting developments in the field of biological physics in recent years is the ability to manipulate single molecules and probe their properties and function. Since its emergence over two decades ago, single molecule force spectroscopy has become a powerful tool to explore the response of biological molecules, including proteins, DNA, RNA and their complexes, to the application of an applied force. The force versus extension response of molecules can provide valuable insight into its mechanical stability, as well as details of the underlying energy landscape. In this review we will introduce the technique of single molecule force spectroscopy using the atomic force microscope (AFM), with particular focus on its application to study proteins. We will review the models which have been developed and employed to extract information from single molecule force spectroscopy experiments. Finally, we will end with a discussion of future directions in this field

The hierarchical emergence of worm-like chain behaviour from globular domain polymer chains

Biological organisms make use of hierarchically organised structures to modulate mechanical behaviour across multiple lengthscales, allowing microscopic objects to generate macroscopic effects. Within these structural hierarchies, the resultant physical behaviour of the entire system is determined not only by the intrinsic mechanical properties of constituent subunits, but also by their organisation in three-dimensional space. When these subunits are polyproteins, colloidal chains or other globular domain polymers, the Kratky–Porod model is often assumed for the individual subunits. Hence, it is implicitly asserted that the polymeric object has an intrinsic parameter, the persistence length, that defines its flexibility. However, the persistence lengths extracted from experiment vary, and are often relatively small. Through a series of simulations on polymer chains formed of globular subunits, we show that the persistence length itself is a hierarchical structural property, related not only to the intrinsic mechanical properties of the underlying monomeric subunits, but emerging due to the organisation of inhomogenous geometry along the polymer contour

Biomolecular self-assembly under extreme Martian mimetic conditions

The recent discovery of subsurface water on Mars has challenged our understanding of the natural limits of life. The presence of magnesium perchlorate (Mg(ClO4)2) on the Martian surface raises the possibility that it may also be present in this subsurface lake. Given that the subsurface lakes on Earth, such as Lake Vostok and Lake Whillans, are capable of harbouring surprising amounts of life, these new findings raise interesting possibilities for how biomolecules might self-assemble in this environment on Mars. Here we investigate the self-association and hydration of the amino acid glycine in aqueous Mg(ClO4)2 at 25°C and −20°C using neutron diffraction with hydrogen isotope substitution and subsequent analysis with empirical potential structure refinement to yield a simulated box of atoms consistent with the scattering data. We find that although the highly chaotropic properties of Mg(ClO4)2 disrupt the hydration and hydrogen bonding ability of the amino acid, as well as the bulk water structure, glycine molecules are nonetheless still able to self-associate. This occurs more readily at lower temperature, where clusters of up to three molecules are observed, allowing us to speculate that the formation of biological molecules is possible in the Martian environment

Spitz and wingless, emanating from distinct borders, cooperate to establish cell fate across the engrailed domain in the drosophila epidermis

A key step in development is the establishment of cell type diversity across a cellular field. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for this process. At each parasegment boundary, cells expressing the Wnt family member Wingless confront cells expressing the homeoprotein Engrailed. The Engrailed-expressing cells normally differentiate as one of two alternative cell types. In investigating the generation of this cell type diversity among the 2-cell-wide Engrailed stripe, we previously showed that Wingless, expressed just anterior to the Engrailed cells, is essential for the specification of anterior Engrailed cell fate. In a screen for additional mutations affecting Engrailed cell fate, we identified anterior open/yan, a gene encoding an inhibitory ETS-domain transcription factor that is negatively regulated by the Rasl-MAP kinase signaling cascade. We find that Anterior Open must be inactivated for posterior Engrailed cells to adopt their correct fate. This is achieved by the EGF receptor (DER), which is required autonomously in the Engrailed cells to trigger the Ras1-MAP kinase pathway. Localized activation of DER is accomplished by restricted processing of the activating ligand, Spitz. Processing is confined to the cell row posterior to the Engrailed domain by the restricted expression of Rhomboid. These cells also express the inhibitory ligand Argos, which attenuates the activation of DER in cell rows more distant from the ligand source. Thus, distinct signals flank each border of the Engrailed domain, as Wingless is produced anteriorly and Spitz posteriorly. Since we also show that En cells have the capacity to respond to either Wingless or Spitz, these cells must choose their fate depending on the relative level of activation of the two pathways.Louise O’Keefe, Scott T. Dougan, Limor Gabay, Erez Raz, Ben-Zion Shilo and Stephen DiNard

Highly compressed water structure observed in a perchlorate aqueous solution

The discovery by the Phoenix Lander of calcium and magnesium perchlorates in Martian soil samples has fueled much speculation that flows of perchlorate brines might be the cause of the observed channeling and weathering in the surface. Here, we study the structure of a mimetic of Martian water, magnesium perchlorate aqueous solution at its eutectic composition, using neutron diffraction in combination with hydrogen isotope labeling and empirical potential structure refinement. We find that the tetrahedral structure of water is heavily perturbed, the effect being equivalent to pressurizing pure water to pressures of order 2 GPa or more. The Mg2+ and ClO4− ions appear charge-ordered, confining the water on length scales of order 9 Å, preventing ice formation at low temperature. This may explain the low evaporation rates and high deliquescence of these salt solutions, which are essential for stability within the low relative humidity environment of the Martian atmosphere

Single molecule protein stabilisation translates to macromolecular mechanics of a protein network

Folded globular proteins are attractive building blocks for biopolymer-based materials, as their mechanically resistant structures carry out diverse biological functionality. While much is now understood about the mechanical response of single folded proteins, a major challenge is to understand and predictably control how single protein mechanics translates to the collective response of a network of connected folded proteins. Here, by utilising the binding of maltose to hydrogels constructed from photo-chemically crosslinked maltose binding protein (MBP), we investigate the effects of protein stabilisation at the molecular level on the macroscopic mechanical and structural properties of a protein-based hydrogel. Rheological measurements show an enhancement in the mechanical strength and energy dissipation of MBP hydrogels in the presence of maltose. Circular dichroism spectroscopy and differential scanning calorimetry measurements show that MBP remains both folded and functional in situ. By coupling these mechanical measurements with mesoscopic structural information obtained by small angle scattering, we propose an occupation model in which higher proportions of stabilised, ligand occupied, protein building blocks translate their increased stability to the macroscopic properties of the hydrogel network. This provides powerful opportunities to exploit environmentally responsive folded protein-based biomaterials for many broad applications

Hierarchical biomechanics: student engagement activities with a focus on biological physics

Hierarchical structure and mechanics are crucial in biological systems as they allow for smaller molecules, such as proteins and sugars, to be used in the construction of large scale biological structures exhibiting properties such as structural support functionality. By exploring the fundamental principles of structure and mechanics at the macroscale, this general theme provides a clear insight into how physics can be applied to the complex questions of biology. With a focus on biopolymer networks and hydrogels, we present a series of interactive activities which cover a range of biophysical concepts at an introductory level, such as viscoelasticity, biological networks and ultimately, hierarchical biomechanics. These activities enable us to discuss multidisciplinary science with a general audience and, given the current trends of research science, this conceptualisation of science is vital for the next generation of scientists

Transnuclear TRP1-Specific CD8 T Cells with High or Low Affinity TCRs Show Equivalent Antitumor Activity

We have generated, via somatic cell nuclear transfer, two independent lines of transnuclear (TN) mice, using as nuclear donors CD8 T cells, sorted by tetramer staining, that recognize the endogenous melanoma antigen TRP1. These two lines of nominally identical specificity differ greatly in their affinity for antigen (TRP1(high) or TRP1(low)) as inferred from tetramer dissociation and peptide responsiveness. Ex vivo-activated CD8 T cells from either TRP1(high) or TRP1(low) mice show cytolytic activity in 3D tissue culture and in vivo, and slow the progression of subcutaneous B16 melanoma. Although naïve TRP1(low) CD8 T cells do not affect tumor growth, upon activation these cells function indistinguishably from TRP1(high) cells in vivo, limiting tumor cell growth and increasing mouse survival. The anti-tumor effect of both TRP1(high) and TRP1(low) CD8 T cells is enhanced in RAG-deficient hosts. However, tumor outgrowth eventually occurs, likely due to T cell exhaustion. The TRP1 TN mice are an excellent model for examining the functional attributes of T cells conferred by TCR affinity, and they may serve as a platform for screening immunomodulatory cancer therapies