Wednesday, July 29, 2015

Ligand-Induced Changes of the Apparent Transition-State Position in Mechanical Protein Unfolding

Johannes Stigler, Matthias Rief

Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.


Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament

Salavat R. Nabiev, Denis A. Ovsyannikov, Galina V. Kopylova, Daniil V. Shchepkin, Alexander M. Matyushenko, Natalia A. Koubassova, Dmitrii I. Levitsky, Andrey K. Tsaturyan, Sergey Y. Bershitsky

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.


Titin Domains Progressively Unfolded by Force Are Homogenously Distributed along the Molecule

Pasquale Bianco, Zsolt Mártonfalvi, Katalin Naftz, Dorina Kőszegi, Miklós Kellermayer

Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecule's length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions.


Monday, July 27, 2015

Controlled translocation of DNA through nanopores in carbon nano-, silicon-nitride- and lipid-coated membranes

Andy Sischka, Lukas Galla, Andreas J. Meyer, Andre Spiering, Sebastian Knust, Michael Mayer, Adam R. Hall, André Beyer, Peter Reimann, Armin Gölzhäuser and Dario Anselmetti

We investigated experimentally and theoretically the translocation forces when a charged polymer is threaded through a solid-state nanopore and found distinct dependencies on the nanopore diameter as well as on the nano membrane material chemistry. For this purpose we utilized dedicated optical tweezers force mechanics capable of probing the insertion of negatively charged double-stranded DNA inside a helium-ion drilled nanopore. We found that both the diameter of the nanopore and the membrane material itself have significant influences on the electroosmotic flow through the nanopore and thus on the threading force. Compared to a bare silicon-nitride membrane, the threading of DNA through only 3 nm thin carbon nano membranes as well as lipid bilayer-coated nanopores increased the threading force by 15% or 85%, respectively. This finding was quantitatively described by our recently developed theoretical model that also incorporates hydrodynamic slip effects on the translocating DNA molecule and the force dependence on the membrane thickness. The additional measurements presented in this paper further support our model.


The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics

Andreas S. Biebricher, Iddo Heller, Roel F. H. Roijmans, Tjalle P. Hoekstra, Erwin J. G. Peterman & Gijs J. L. Wuite

DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy.


Information and thermodynamics: experimental verification of Landauer's Erasure principle

Antoine Bérut, Artyom Petrosyan and Sergio Ciliberto

We present an experiment in which a one-bit memory is constructed, using a system of a single colloidal particle trapped in a modulated double-well potential. We measure the amount of heat dissipated to erase a bit and we establish that in the limit of long erasure cycles the mean dissipated heat saturates at the Landauer bound, i.e. the minimal quantity of heat necessarily produced to delete a classical bit of information. This result demonstrates the intimate link between information theory and thermodynamics. To stress this connection we also show that a detailed Jarzynski equality is verified, retrieving the Landauer's bound independently of the work done on the system. The experimental details are presented and the experimental errors carefully discussed.

Wednesday, July 22, 2015

Nanochannel Electroporation as a Platform for Living Cell Interrogation in Acute Myeloid Leukemia

Xi Zhao, Xiaomeng Huang, Xinmei Wang, Yun Wu, Ann-Kathrin Eisfeld, Sebastian Schwind, Daniel Gallego-Perez, Pouyan E. Boukany, Guido I. Marcucci and Ly James Lee

A comprehensive micro/nanofluidics platform for single-cell analysis based on nanochannel electroporation (NEP) and molecular beacon (MB) is presented in this study. The platform can quantitatively analyze multiple RNA species in individual cells with minimal cell damage. Furthermore, it is capable of delivering nucleic acids into target cells and subsequently detecting their responses at RNA level, e.g., microRNA (miRNA). It is known that as the downstream targets of miR-29b, DNMT3A/B can be downregulated by miR-29b overexpression. To demonstrate the activity of delivered miR-29b by NEP and the analytical function of the platform, the decreased expression of DNMT3A/B in acute myeloid leukemia (AML) cells was verified at single-cell level by simultaneous detection of multiple genes in the same cell. The potential of such platform on intracellular pathway studies has also been explored by investigating the upregulation efficiencies of miR-181a through different pathways in AML cells. The results showed that an indirect approach by C/EBPα-p30 peptide expression would have a stronger effect than direct transfection of the miR-181a gene. The platform has also shown its advantages over established technologies in the analysis of cells that are hard to transfect.


Advances in the measurement of red blood cell deformability: A brief review

Kim, Jeongho | Lee, HoYoon | Shin, Sehyun

Red blood cells (RBCs) exhibit a unique deformability, which enables them to change shape reversibly in response to an external force. The deformability of RBCs allows them to flow in microvessels while transporting oxygen and carbon dioxide. In this review, we discussed the major determinants of RBC deformability, which include cell geometry, internal viscosity, rheological properties of the membrane, osmotic pressure, calcium, nitric oxide, temperature, ageing, and depletion of adenosine triphosphate. Additionally, we highlighted the various methods and techniques used to measure RBC deformability. Individual cell analyses (pipette aspiration and optical tweezers) and bulk cell analyses (ektacytometry, multiple channels) were described and compared. Finally, we reviewed the correlation between RBC deformability and clinical outcomes such as diabetic microangiopathy.


Probing the structural dynamics of proteins and nucleic acids with optical tweezers

Dustin B Ritchie, Michael T Woodside

Conformational changes are an essential feature of most molecular processes in biology. Optical tweezers have emerged as a powerful tool for probing conformational dynamics at the single-molecule level because of their high resolution and sensitivity, opening new windows on phenomena ranging from folding and ligand binding to enzyme function, molecular machines, and protein aggregation. By measuring conformational changes induced in a molecule by forces applied by optical tweezers, new insight has been gained into the relationship between dynamics and function. We discuss recent advances from studies of how structure forms in proteins and RNA, including non-native structures, fluctuations in disordered proteins, and interactions with chaperones assisting native folding. We also review the development of assays probing the dynamics of complex protein–nucleic acid and protein–protein assemblies that reveal the dynamic interactions between biomolecular machines and their substrates.


Monday, July 20, 2015

Curved singular beams for three-dimensional particle manipulation

Juanying Zhao, Ioannis D. Chremmos, Daohong Song, Demetrios N. Christodoulides, Nikolaos K. Efremidis & Zhigang Chen

For decades, singular beams carrying angular momentum have been a topic of considerable interest. Their intriguing applications are ubiquitous in a variety of fields, ranging from optical manipulation to photon entanglement, and from microscopy and coronagraphy to free-space communications, detection of rotating black holes, and even relativistic electrons and strong-field physics. In most applications, however, singular beams travel naturally along a straight line, expanding during linear propagation or breaking up in nonlinear media. Here, we design and demonstrate diffraction-resisting singular beams that travel along arbitrary trajectories in space. These curved beams not only maintain an invariant dark “hole” in the center but also preserve their angular momentum, exhibiting combined features of optical vortex, Bessel, and Airy beams. Furthermore, we observe three-dimensional spiraling of microparticles driven by such fine-shaped dynamical beams. Our findings may open up new avenues for shaped light in various applications.