Zhi Qi, Robert A Pugh, Maria Spies, Yann R Chemla
Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones.
DOI
Tuesday, February 4, 2014
Discriminatory optical force for chiral molecules
Robert P Cameron, Stephen M Barnett and Alison M Yao
We suggest that the force F exerted upon a chiral molecule by light assumes the form $\mathbf {F}=a\boldsymbol {\nabla } w+b\boldsymbol {\nabla } h$ under appropriate circumstances, where a and b pertain to the molecule whilst w and h are the local densities of electric energy and helicity in the optical field; the gradients $\boldsymbol {\nabla }$ of these quantities thus governing the molecule's centre-of-mass motion. Whereas a is identical for the mirror-image forms or enantiomers of the molecule, b has opposite signs; the associated contribution to F therefore pointing in opposite directions. A simple optical field is presented for which $\boldsymbol {\nabla } w$ vanishes but $\boldsymbol {\nabla }h$ does not, so that F is absolutely discriminatory. We then present two potential applications: a Stern–Gerlach-type deflector capable of spatially separating the enantiomers of a chiral molecule and a diffraction grating to which chiral molecules alone are sensitive; the resulting diffraction patterns thus encoding information about their chiral geometry.
DOI
We suggest that the force F exerted upon a chiral molecule by light assumes the form $\mathbf {F}=a\boldsymbol {\nabla } w+b\boldsymbol {\nabla } h$ under appropriate circumstances, where a and b pertain to the molecule whilst w and h are the local densities of electric energy and helicity in the optical field; the gradients $\boldsymbol {\nabla }$ of these quantities thus governing the molecule's centre-of-mass motion. Whereas a is identical for the mirror-image forms or enantiomers of the molecule, b has opposite signs; the associated contribution to F therefore pointing in opposite directions. A simple optical field is presented for which $\boldsymbol {\nabla } w$ vanishes but $\boldsymbol {\nabla }h$ does not, so that F is absolutely discriminatory. We then present two potential applications: a Stern–Gerlach-type deflector capable of spatially separating the enantiomers of a chiral molecule and a diffraction grating to which chiral molecules alone are sensitive; the resulting diffraction patterns thus encoding information about their chiral geometry.
DOI
Membrane Shape Modulates Transmembrane Protein Distribution
Sophie Aimon, Andrew Callan-Jones, Alice Berthaud, Mathieu Pinot, Gilman E.S. Toombes, Patricia Bassereau
Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins.
DOI
Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins.
DOI
Monday, February 3, 2014
Mobility Analysis of Super-Resolved Proteins on Optically Stretched DNA: Comparing Imaging Techniques and Parameters
Dr. Iddo Heller, Gerrit Sitters, Onno D. Broekmans, Dr. Andreas S. Biebricher, Prof. Dr. Gijs J. L. Wuite, Prof. Dr. Erwin J. G. Peterman
Fluorescence microscopy in conjunction with optical tweezers is well suited to the study of protein mobility on DNA. Here, we evaluate the benefits and drawbacks of super-resolution and conventional imaging techniques for the analysis of one-dimensional (1D) protein diffusion as commonly observed for DNA-binding proteins. In particular, we demonstrate the visualization of DNA-bound proteins using wide-field, confocal, and stimulated emission depletion (STED) microscopy. We review the suitability of these techniques to conditions of high protein density, and quantify their performance in terms of spatial and temporal resolution. Tracking proteins on DNA forces one to make a choice between localization precision on the one hand, and the number and rate of localizations on the other, by altering imaging modality, excitation intensity, and acquisition rate. Using simulated diffusion data, we quantify the effect of these imaging conditions on the accuracy of 1D diffusion analysis. In addition, we consider the case of diffusion confined between local roadblocks, a case particularly relevant for proteins bound to DNA. Together these results provide guidelines that can assist in judiciously optimizing the experimental conditions required for the analysis of protein mobility on DNA and other 1D systems.
DOI
Fluorescence microscopy in conjunction with optical tweezers is well suited to the study of protein mobility on DNA. Here, we evaluate the benefits and drawbacks of super-resolution and conventional imaging techniques for the analysis of one-dimensional (1D) protein diffusion as commonly observed for DNA-binding proteins. In particular, we demonstrate the visualization of DNA-bound proteins using wide-field, confocal, and stimulated emission depletion (STED) microscopy. We review the suitability of these techniques to conditions of high protein density, and quantify their performance in terms of spatial and temporal resolution. Tracking proteins on DNA forces one to make a choice between localization precision on the one hand, and the number and rate of localizations on the other, by altering imaging modality, excitation intensity, and acquisition rate. Using simulated diffusion data, we quantify the effect of these imaging conditions on the accuracy of 1D diffusion analysis. In addition, we consider the case of diffusion confined between local roadblocks, a case particularly relevant for proteins bound to DNA. Together these results provide guidelines that can assist in judiciously optimizing the experimental conditions required for the analysis of protein mobility on DNA and other 1D systems.
DOI
Single Cell Transfection with Single Molecule Resolution Using a Synthetic Nanopore
Volker Kurz , Tetsuya Tanaka , and Gregory Timp
We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient, but also transfects with single molecule resolution at low voltage (1V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precision—no other method offers such capabilities.
DOI
We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient, but also transfects with single molecule resolution at low voltage (1V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precision—no other method offers such capabilities.
DOI
A vertebrate myosin-I structure reveals unique insights into myosin mechanochemical tuning
Henry Shuman, Michael J. Greenberg, Adam Zwolak, Tianming Lin, Charles V. Sindelar, Roberto Dominguez, and E. Michael Ostap
Myosins are molecular motors that power diverse cellular processes, such as rapid organelle transport, muscle contraction, and tension-sensitive anchoring. The structural adaptations in the motor that allow for this functional diversity are not known, due, in part, to the lack of high-resolution structures of highly tension-sensitive myosins. We determined a 2.3-Å resolution structure of apo-myosin-Ib (Myo1b), which is the most tension-sensitive myosin characterized. We identified a striking unique orientation of structural elements that position the motor’s lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. Single-molecule and biochemical analyses show that the N terminus plays an important role in stabilizing the post power-stroke conformation of Myo1b and in tuning the rate of the force-sensitive transition. We propose that this region plays a general role in tuning the mechanochemical properties of myosins.
Myosins are molecular motors that power diverse cellular processes, such as rapid organelle transport, muscle contraction, and tension-sensitive anchoring. The structural adaptations in the motor that allow for this functional diversity are not known, due, in part, to the lack of high-resolution structures of highly tension-sensitive myosins. We determined a 2.3-Å resolution structure of apo-myosin-Ib (Myo1b), which is the most tension-sensitive myosin characterized. We identified a striking unique orientation of structural elements that position the motor’s lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. Single-molecule and biochemical analyses show that the N terminus plays an important role in stabilizing the post power-stroke conformation of Myo1b and in tuning the rate of the force-sensitive transition. We propose that this region plays a general role in tuning the mechanochemical properties of myosins.
Trap position control in the vicinity of reflecting surfaces in optical tweezers
D. A. Shilkin, E. V. Lyubin, I. V. Soboleva, A. A. Fedyanin
Shift of the trap position from the laser beam waist of optical tweezers is studied experimentally in the presence of a reflecting surface in the vicinity of the focal plane. A standing wave is formed owing to the interference of waves forming the waist and reflected from the surface. The standing wave is shown to affect significantly the resulting trap position. The distance between the surface and the stable optical trap as a function of the trapped particle size is studied numerically. A new method to stabilize the position of the microparticle relative to the surface is proposed. The localization accuracy is determined by the Brownian fluctuations in optical tweezers and is about 10 nm for effective trap stiffness of 4 × 10−5 N/m.
DOI
Shift of the trap position from the laser beam waist of optical tweezers is studied experimentally in the presence of a reflecting surface in the vicinity of the focal plane. A standing wave is formed owing to the interference of waves forming the waist and reflected from the surface. The standing wave is shown to affect significantly the resulting trap position. The distance between the surface and the stable optical trap as a function of the trapped particle size is studied numerically. A new method to stabilize the position of the microparticle relative to the surface is proposed. The localization accuracy is determined by the Brownian fluctuations in optical tweezers and is about 10 nm for effective trap stiffness of 4 × 10−5 N/m.
DOI
Single-Molecule Measurements of the CCR5 mRNA Unfolding Pathways
Michel de Messieres, Jen-Chien Chang, Ashton Trey Belew, Arturas Meskauskas, Jonathan D. Dinman, Arthur La Porta
Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown. Here we employ optical trapping techniques to investigate the structure of a −1 programmed ribosomal frameshift (−1 PRF) sequence element located in the CCR5 mRNA, which encodes a coreceptor for HIV-1 and is, to our knowledge, the first known human −1 PRF signal of nonviral origin. We begin by presenting a set of computationally predicted structures that include pseudoknots. We then employ what we believe to be new analytical techniques for measuring the effective free energy landscapes of biomolecules. We find that the −1 PRF element manifests several distinct unfolding pathways when subject to end-to-end force, one of which is consistent with a proposed pseudoknot conformation, and another of which we have identified as a folding intermediate. The dynamic ensemble of conformations that CCR5 mRNA exhibits in the single-molecule experiments may be a significant feature of the frameshifting mechanism.
DOI
Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown. Here we employ optical trapping techniques to investigate the structure of a −1 programmed ribosomal frameshift (−1 PRF) sequence element located in the CCR5 mRNA, which encodes a coreceptor for HIV-1 and is, to our knowledge, the first known human −1 PRF signal of nonviral origin. We begin by presenting a set of computationally predicted structures that include pseudoknots. We then employ what we believe to be new analytical techniques for measuring the effective free energy landscapes of biomolecules. We find that the −1 PRF element manifests several distinct unfolding pathways when subject to end-to-end force, one of which is consistent with a proposed pseudoknot conformation, and another of which we have identified as a folding intermediate. The dynamic ensemble of conformations that CCR5 mRNA exhibits in the single-molecule experiments may be a significant feature of the frameshifting mechanism.
DOI
Optical tweezers reveal force plateau and internal friction in PEG-induced DNA condensation
Heikki Ojala, Gabija Ziedaite, Anders E. Wallin, Dennis H. Bamford, Edward Hæggström
The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 μm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force–extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force–extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.
DOI
The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 μm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force–extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force–extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.
DOI
Diverse Metastable Structures Formed by Small Oligomers of α-Synuclein Probed by Force Spectroscopy
Krishna Neupane, Allison Solanki, Iveta Sosova, Miro Belov, Michael T. Woodside
Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson’s disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.
DOI
Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson’s disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.
DOI
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