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Single molecule biophysics

Joerg Schnitzbauer, Yina Wang, Shijie Zhao, Matthew Bakalar, Tulip Nuwal, Baohui Chen, Bo Huang
Superresolution images reconstructed from single-molecule localizations can reveal cellular structures close to the macromolecular scale and are now being used routinely in many biomedical research applications. However, because of their coordinate-based representation, a widely applicable and unified analysis platform that can extract a quantitative description and biophysical parameters from these images is yet to be established. Here, we propose a conceptual framework for correlation analysis of coordinate-based superresolution images using distance histograms...
March 12, 2018: Proceedings of the National Academy of Sciences of the United States of America
Stephan Block, Srdjan S Aćimović, Nils Odebo Länk, Mikael Käll, Fredrik Höök
Fluorescence correlation spectroscopy (FCS) has provided a wealth of information on the composition, structure, and dynamics of cell membranes. However, it has proved challenging to reach the spatial resolution required to resolve biophysical interactions at the nm-scale relevant to many crucial membrane processes. In this work, we form artificial cell membranes on dimeric, nanoplasmonic antennas, which shrink the FCS probe volume down to the ~20 nm length-scale. By analysing the autocorrelation functions (ACFs) associated with the fluorescence bursts from individual fluorescently tagged lipids moving through the antenna "hot spots", we show that the confinement of the optical readout volume below the diffraction limit allows the temporal resolution of FCS to be increased by up to 3 orders of magnitude...
March 12, 2018: ACS Nano
Quinn Li, Laura Folly da Silva Constantino, M Ashley Spies
Discovery of novel tool compounds and drug leads against a range of unorthodox protein targets has pushed both experimental screening methodologies as well as the field of structure-based design to the limit in recent years. Increasingly, it has been recognized that some of the most desirable targets for the development of small-molecule effectors are actually protein-protein and protein-nucleic acid interactions. There are numerous nontrivial challenges to pursuing small-molecule lead compounds directed toward PPIs and PNIs: relatively shallow cavities, large surface areas that are natively complexed to macromolecules, complex patterns of interstitial waters, a paucity of "hot spots," large conformational changes upon ligand binding, etc...
2018: Methods in Enzymology
Rikiya Watanabe, Takaharu Sakuragi, Hiroyuki Noji, Shigekazu Nagata
Transmembrane protein 16F (TMEM16F) is a Ca2+ -dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro...
March 5, 2018: Proceedings of the National Academy of Sciences of the United States of America
Jianxin Guan, Chuancheng Jia, Yanwei Li, Zitong Liu, Jinying Wang, Zhongyue Yang, Chunhui Gu, Dingkai Su, Kendall N Houk, Deqing Zhang, Xuefeng Guo
Single-molecule detection can reveal time trajectories and reaction pathways of individual intermediates/transition states in chemical reactions and biological processes, which is of fundamental importance to elucidate their intrinsic mechanisms. We present a reliable, label-free single-molecule approach that allows us to directly explore the dynamic process of basic chemical reactions at the single-event level by using stable graphene-molecule single-molecule junctions. These junctions are constructed by covalently connecting a single molecule with a 9-fluorenone center to nanogapped graphene electrodes...
February 2018: Science Advances
Diana M Mitrea, Jaclyn A Cika, Christopher B Stanley, Amanda Nourse, Paulo L Onuchic, Priya R Banerjee, Aaron H Phillips, Cheon-Gil Park, Ashok A Deniz, Richard W Kriwacki
Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid-liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (R-motifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions...
February 26, 2018: Nature Communications
Philip C Nelson
Resonance energy transfer has become an indispensable experimental tool for single-molecule and single-cell biophysics. Its physical underpinnings, however, are subtle: it involves a discrete jump of excitation from one molecule to another, and so we regard it as a strongly quantum-mechanical process. And yet its kinetics differ from what many of us were taught about two-state quantum systems, quantum superpositions of the states do not seem to arise, and so on. Although J. R. Oppenheimer and T. Förster navigated these subtleties successfully, it remains hard to find an elementary derivation in modern language...
February 16, 2018: Biophysical Journal
Shyamal Subramanyam, Maria Spies
The RAD51 DNA strand exchange protein plays an important role in maintaining the integrity of the human genome. It promotes homology-directed DNA repair by exchanging strands between the damaged and the intact DNA molecules. It also plays an important role in stabilizing distressed DNA replication forks. When overexpressed or misregulated, however, RAD51 contributes to "rogue," genome destabilizing events that can lead to cancer, cell death, and to acquisition of chemotherapy resistance by cancerous cells...
2018: Methods in Enzymology
Nayan P Agarwal, Michael Matthies, Bastian Joffroy, Thorsten L Schmidt
The programmability of DNA enables constructing nanostructures with almost any arbitrary shape, which can be decorated with many functional materials. Moreover, dynamic structures can be realized such as molecular motors and walkers. In this work, we have explored the possibility to synthesize the complementary sequences to single-stranded gap regions in the DNA origami scaffold cost effectively by a DNA polymerase rather than by a DNA synthesizer. For this purpose, four different wireframe DNA origami structures were designed to have single-stranded gap regions...
February 16, 2018: ACS Nano
Susan P Gilbert, Stephanie Guzik-Lendrum, Ivan Rayment
Kinesin-2s are major transporters of cellular cargoes. This subfamily contains both homodimeric kinesins whose catalytic domains result from the same gene product and heterodimeric kinesins with motor domains derived from two different gene products. In this review, we focus on the progress to define the biochemical and biophysical properties of the kinesin-2 family members. Our understanding of their mechanochemical capabilities has been advanced by the ability to identify the kinesin-2 genes in multiple species, expression and purification of these motors for single molecule and ensemble assays, and development of new technologies enabling quantitative measurements of kinesin activity with greater sensitivity...
February 14, 2018: Journal of Biological Chemistry
Markus Götz, Philipp Wortmann, Sonja Schmid, Thorsten Hugel
Single-molecule Förster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM)...
January 30, 2018: Journal of Visualized Experiments: JoVE
Tamara Šmidlehner, Ivo Piantanida, Gennaro Pescitelli
The structural characterization of non-covalent complexes between nucleic acids and small molecules (ligands) is of a paramount significance to bioorganic research. Highly informative methods about nucleic acid/ligand complexes such as single crystal X-ray diffraction or NMR spectroscopy cannot be performed under biologically compatible conditions and are extensively time consuming. Therefore, in search for faster methods which can be applied to conditions that are at least similar to the naturally occurring ones, a set of polarization spectroscopy methods has shown highly promising results...
2018: Beilstein Journal of Organic Chemistry
Diana Di Paolo, Richard M Berry
For the last 2 decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signaling in E. coli, including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical. For example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart. FPs may interfere with the native interactions of the protein, and their chromophores have low brightness and photostability, and fast photobleaching rates...
2018: Methods in Molecular Biology
B C Publio, T A Moura, C H M Lima, M S Rocha
We have performed a biophysical characterization, at single molecule level, of the interaction between the DNA molecule and the biogenic polyamine putrescine. By using force spectroscopy, we were able to monitor the complexes formation as putrescine is added to the sample, determining the mechanical properties of such complexes and the physicochemical (binding) parameters of the interaction for three different ionic strengths. In particular, it was shown that the behavior of the equilibrium binding constant as a function of the counterion concentration deviates from the prediction of the Record-Lohman model...
January 31, 2018: International Journal of Biological Macromolecules
Taylor Firman, Stephen Wedekind, Thomas J McMorrow, Kingshuk Ghosh
Gene networks with feedback often involve interactions between multiple species of bio-molecules, much more than experiments can actually monitor. Coupled with this is the challenge that experiments often measure gene expression in noisy fluorescence instead of protein numbers. How do we infer biophysical information and characterize the underlying circuits from this limited and convoluted data? We address this by building stochastic models using the principle of Maximum Caliber (MaxCal). MaxCal uses the basic information of synthesis, degradation, and feedback -- without invoking any other auxiliary species and ad hoc reactions -- to generate stochastic trajectories similar to those typically measured in experiments...
February 6, 2018: Journal of Physical Chemistry. B
Colin D Kinz-Thompson, Ruben L Gonzalez
Many time-resolved single-molecule biophysics experiments seek to characterize the kinetics of biomolecular systems exhibiting dynamics that challenge the time resolution of the given technique. Here, we present a general, computational approach to this problem that employs Bayesian inference to learn the underlying dynamics of such systems, even when they are much faster than the time resolution of the experimental technique being used. By accurately and precisely inferring rate constants, our Bayesian inference for the analysis of subtemporal resolution dynamics approach effectively enables the experimenter to super-resolve the poorly resolved dynamics that are present in their data...
January 23, 2018: Biophysical Journal
C Schaefer
Fluids with spatial density variations of single or mixed molecules play a key role in biophysics, soft matter, and materials science. The fluid structures usually form via spinodal decomposition or nucleation following an instantaneous destabilization of the initially disordered fluid. However, in practice, an instantaneous quench is often not viable, and the rate of destabilization may be gradual rather than instantaneous. In this work we show that the commonly used phenomenological descriptions of fluid structuring are inadequate under these conditions...
January 19, 2018: Physical Review Letters
Suman Das, Adam Eisen, Yi-Hsuan Lin, Hue Sun Chan
In view of recent intense experimental and theoretical interests in the biophysics of liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs), heteropolymer models with chain molecules configured as self-avoiding walks on the simple cubic lattice are constructed to study how phase behaviors depend on the sequence of monomers along the chains. To address pertinent general principles, we focus primarily on two fully charged 50-monomer sequences with significantly different charge patterns...
February 3, 2018: Journal of Physical Chemistry. B
Alexander Gust, Leonhard Jakob, Daniela Zeitler, Astrid Bruckmann, Kevin Kramm, Sarah Willkomm, Philip Tinnefeld, Gunter Meister, Dina Grohmann
Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states and modifications like phosphorylation affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single-molecule FRET measurements allow the exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome as a method for the site-specific coupling of organic dyes into native, non-recombinant mammalian proteins is lacking...
February 2, 2018: Chembiochem: a European Journal of Chemical Biology
Ivan Rey, David A Garcia, Brittany A Wheatley, Wenxia Song, Arpita Upadhyaya
Cells of the adaptive immune system recognize pathogenic peptides through specialized receptors on their membranes. The engagement of these receptors with antigen leads to cell activation, which induces profound changes in the cell including cytoskeleton remodeling and membrane deformation. During this process, receptors and signaling molecules undergo spatiotemporal reorganization to form signaling microclusters and the immunological synapse. The cytoskeletal and membrane dynamics also leads to exertion of forces on the cell-substrate interface...
2018: Methods in Molecular Biology
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