Friday, September 25, 2009

Proofreading dynamics of a processive DNA polymerase

Borja Ibarra, Yann R Chemla, Sergey Plyasunov, Steven B Smith, José M Lázaro, Margarita Salas and Carlos Bustamante

Replicative DNA polymerases present an intrinsic proofreading activity during which the DNA primer chain is transferred between the polymerization and exonuclease sites of the protein. The dynamics of this primer transfer reaction during active polymerization remain poorly understood. Here we describe a single-molecule mechanical method to investigate the conformational dynamics of the intramolecular DNA primer transfer during the processive replicative activity of the Phi 29 DNA polymerase and two of its mutants. We find that mechanical tension applied to a single polymerase-DNA complex promotes the intramolecular transfer of the primer in a similar way to the incorporation of a mismatched nucleotide. The primer transfer is achieved through two novel intermediates, one a tension-sensitive and functional polymerization conformation and a second non-active state that may work as a fidelity check point for the proofreading reaction.

Tuesday, September 22, 2009

Flatness-based approach for the manipulation of a microscopic particle by optical tweezers

Carlos Aguilar-Ibanez, Armando Barranon Cedillo, Hebertt Sira-Ramirez, and Luis I. Rosas-Soriano

Based on the fact that optical tweezers (OT) constitute a flat system, with flat outputs given by the horizontal and vertical position coordinates of the geometric centre of the laser beam, a flatness-based control strategy for the manipulation of a microscopical particle is presented in this article. The control strategy is derived under the assumption that the particle is suspended in a frictionless medium, obtaining a simple controller with a relatively simple stability analysis. The control strategy is tested by tracking a straight line, an elliptic curve, and carrying out the rest-to-rest transfer manoeuvre task by using a smooth trajectory.

Characterization of single heat-activated Bacillus spores using laser tweezers 
Raman spectroscopy

Pengfei Zhang, Peter Setlow, and Yongqing Li

Heat activation of dormant bacterial spores is a short treatment at a sublethal temperature that potentiates and synchronizes spore germination. In this paper, laser tweezers Raman spectroscopy (LTRS) was used to study the heat activation of single spores of Bacillus cereus and Bacillus subtilis. We measured the Raman spectra of single spores without treatment, during heat activation at 65 °C (B. cereus) or 70 °C (B. subtilis), and following heat activation and cooling to 25 °C. Principle component analysis (PCA) was applied to discriminate among the three groups of spores based on their Raman spectra. The results indicated that: (1) there are large changes in the Raman bands of Ca-DPA and protein for both B. cereus and B. subtilis spores during heat activation, indicative of changes in spore core state and partial protein denaturation at the heat activation temperatures; (2) these spectral changes become smaller once the heated spores are cooled, consistent with heat activation being reversible; (3) minor spectral differences between untreated and heat-activated and cooled spores can be discriminated by PCA based on non-polarized and polarized Raman spectra; and (4) analysis based on polarized Raman spectra reveals that partial denaturation of protein during heat activation is mainly observed in the vertically polarized component.

Friday, September 18, 2009

Mechanical properties of a giant liposome studied using optical tweezers

Yoko Shitamichi, Masatoshi Ichikawa and Yasuyuki Kimura

The mechanical properties of a micrometer-sized giant liposome are studied by deforming it from the inside using dual-beam optical tweezers. As the liposome is extended, its shape changes from a sphere to a lemon shape, and finally, a tubular part is generated. The surface tension σ and the bending rigidity κ of the lipid membrane are obtained from the measured force–extension curve. In a one-phase liposome, it was found that σ increases as the charged component increases but κ remains approximately constant. In a two-phase liposome, the characteristic deformation and the force–extension curve differ from those observed for the one-phase liposome.

Pattern switching and polarizability for colloids in optical-trap arrays

C. Reichhardt and C. J. Olson Reichhardt

We show that colloidal molecular crystal states interacting with a periodic substrate, such as an optical-trap array, and a rotating external field can undergo a rapid pattern switching in which the orientation of the crystal changes. In some cases, a martensiticlike symmetry switching occurs. It is also possible to create a polarized state where the colloids in each substrate minimum develop a director field which smoothly rotates with the external drive, similar to liquid-crystal behavior. These results open the possibility for creating different types of devices using photonic band-gap materials, and should be generalizable to a variety of other condensed matter systems with multiple particle trapping.

Subdiffusive behavior in a trapping potential: Mean square displacement and velocity autocorrelation function

M. A. Despósito and A. D. Viñales

A theoretical framework for analyzing stochastic data from single-particle tracking in viscoelastic materials and under the influence of a trapping potential is presented. Starting from a generalized Langevin equation, we found analytical expressions for the two-time dynamics of a particle subjected to a harmonic potential. The mean-square displacement and the velocity autocorrelation function of the diffusing particle are given in terms of the time lag. In particular, we investigate the subdiffusive case. Using a power-law memory kernel, exact expressions for the mean-square displacement and the velocity autocorrelation function are obtained interms of Mittag-Leffler functions and their derivatives. The behaviors for short-, intermediate-, and long-time lags are investigated in terms of the involved parameters. Finally, the validity of usual approximations is examined.

Entropic boundary effects on the elasticity of short DNA molecules

Yih-Fan Chen, David P. Wilson, Krishnan Raghunathan, and Jens-Christian Meiners

We have measured the entropic elasticity of double-stranded-DNA molecules ranging from 247 to 1298 bp in length using axial force-clamp optical tweezers. We show that entropic end effects and excluded-volume forces from surface attachments become significant for such short molecules. The effective persistence length of the shortest molecules decreases by a factor of 2 compared to the established value for long molecules, and excluded-volume forces extend the molecules to about one third of their nominal contour length. We interpret these results in the framework of an inextensible semiflexible rod model.

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

Hannie S. van der Honing , Norbert C. A. de Ruijter , Anne Mie C. Emons and Tijs Ketelaar

Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

Thursday, September 17, 2009

The elastic properties of the Cryptococcus neoformans capsule

Frases S, Pontes B, Nimrichter L, Rodrigues ML, Viana NB, Casadevall A.

Microbial capsules are important for virulence, but their architecture and physical properties are poorly understood. The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide capsule that is necessary for virulence and is the target of protective antibody responses. To study the C. neoformans capsule we developed what we believe is a new approach whereby we probed the capsular elastic properties by applying forces using polystyrene beads manipulated with optical tweezers. This method allowed us to determine the Young's modulus for the capsule in various conditions that affect capsule growth. The results indicate that the Young's modulus of the capsule decreases with its size and increases with the Ca(2+) concentration in solution. Also, capsular polysaccharide manifests an unexpected affinity for polystyrene beads, a property that may function in attachment to host cells and environmental structures. Bead probing with optical tweezers provides a new, nondestructive method that may have wide applicability for studying the effects of growth conditions, immune components, and drugs on capsular properties.

Optical trapping of quantum dots in a metallic nanotrap

C Dineen, M Reichelt, S W Koch and J V Moloney

We present a novel optical trap structure for the confinement of a freely mobile quantum dot to a sub-diffraction-limited volume. We examine the optical forces on a single quantum dot, under excitonic resonance conditions, in the presence of resonantly enhanced electric fields in the near-field of a suitably engineered metal nanostructure. A numerical scheme to calculate the electromagnetic force on a quantum dot using anadaptive mesh refinement (AMR) FDTD code combined with microscopic material equations is employed. Numerically derived forces on a single quantum dot are presented and it is shown that the dot may be confined in the proposed structure.

Optical Vortices from Liquid Crystal Droplets

Etienne Brasselet, Naoki Murazawa, Hiroaki Misawa, and Saulius Juodkazis

We report on the generation of mono- and polychromatic optical phase singularities from micron-sized birefringent droplets. This is done experimentally by using liquid crystal droplets whose three dimensional architecture of the optical axis is controlled within the bulk by surfactant agents. Because of its microscopic size these optical vortex generators are optically trapped and manipulated at will, thus realizing a robust self-aligned micro-optical device for orbital angular momentum conversion. Experimental observations are supported by a simple model of optical spin-orbit coupling in uniaxial dielectrics that emphasizes the prominent role of thetransverse optical anisotropy with respect to the beam propagation direction.

Mechanical Oscillation and Cooling Actuated by the Optical Gradient Force

Qiang Lin, Jessie Rosenberg, Xiaoshun Jiang, Kerry J. Vahala, and Oskar Painter

In this work, we combine the large per-photon optical gradient force with the sensitive feedback of a high quality factor whispering-gallery microcavity. The cavity geometry, consisting of a pair of silica disks separated by a nanoscale gap, shows extremely strong dynamical backaction, powerful enough to excite coherent oscillations even under heavily damped conditions (mechanical Q
[approximate]4). In vacuum, the threshold for regenerative mechanical oscillation is lowered to an optical input power of only 270 nW, or roughly 1000 stored cavity photons, and efficient cooling of the mechanical motion is obtained with a temperature compression factor of nearly 14 dB with an input optical power of only 11 µW.

Wednesday, September 16, 2009

Controlled assembly of In2O3 nanowires on electronic circuits using scanning optical tweezers

Song-Woo Lee, Gunho Jo, Takhee Lee, and Yong-Gu Lee

In2O3 nanowires can be used effectively as building blocks in the production of electronic circuits used in transparent and flexible electronic devices. The fabrication of these devices requires a controlled assembly of nanowires at crucial places and times. However, this kind of controlled assembly, which results in the fusion of nanowires to circuits, is still very difficult to execute. In this study, we demonstrate the benefits of using various lengths of In2O3 nanowires by using non-contact mechanisms, such as scanning optical tweezers, to place them on designated targets during the fabrication process. Furthermore, these nanowires can be stabilized at both ends of the conducting wires using a focused laser, and later in the process, the annealed technique, so that proper flow of electrons is affected.

ATP-dependent mechanics of red blood cells

Timo Betz, Martin Lenz, Jean-François Joanny and Cécile Sykes

Red blood cells are amazingly deformable structures able to recover their initial shape even after large deformations as when passing through tight blood capillaries. The reason for this exceptional property is found in the composition of the membrane and the membrane-cytoskeleton interaction. We investigate the mechanics and the dynamics of RBCs by a unique noninvasive technique, using weak optical tweezers to measure membrane fluctuation amplitudes with μs temporal and sub nm spatial resolution. This enhanced edge detection method allows to span over >4 orders of magnitude in frequency. Hence, we can simultaneously measure red blood cell membrane mechanical properties such as bending modulusκ = 2.8 ± 0.3 × 10−19J = 67.6 ± 7.2 kBT, tension σ = 6.5 ± 2.1 × 10−7N/m, and an effective viscosity ηeff = 81 ± 3.7 × 10−3 Pa s that suggests unknown dissipative processes. We furthermore show that cell mechanics highly depends on the membrane-spectrin interaction mediated by the phosphorylation of the interconnection protein 4.1R. Inhibition and activation of this phosphorylation significantly affects tension and effective viscosity. Our results show that on short time scales (slower than 100 ms) the membrane fluctuates as in thermodynamic equilibrium. At time scales longer than 100 ms, the equilibrium description breaks down and fluctuation amplitudes are higher by 40% than predicted by the membrane equilibrium theory. Possible explanations for this discrepancy are influences of the spectrin that is not included in the membrane theory or nonequilibrium fluctuations that can be accounted for by defining a nonthermal effective energy of up to Eeff = 1.4 ± 0.1 kBT, that corresponds to an actively increased effective temperature.

Tuesday, September 15, 2009

Optical angular momentum transfer to microrotors fabricated by two-photon photopolymerization

Theodor Asavei, Vincent L Y Loke, Marco Barbieri, Timo A Nieminen, Norman R Heckenberg and Halina Rubinsztein-Dunlop

We design, fabricate and test optically driven microrotors a few microns in size. The rotors are trapped and rotated in optical tweezers using an LG02 Laguerre–Gaussian laser beam. We verify that we can accurately measure the total optical torque by measuring the spin angular momentum transfer for three different polarizations, by comparing the optical torque with the optical torque calculated using computational electrodynamics and the viscous drag torque determined from the rotation rate and computational fluid dynamics. The torque agrees with that expected from the design principles and electromagnetic modelling of the torque within the optical trap.

Particle size limits when using optical trapping and deflection of particles for sorting using diode laser bars

Robert W. Applegate Jr., David W. M. Marr, Jeff Squier, and Steven W. Graves

We explore a simple, inexpensive approach to large particle manipulation using diode laser bar optical trapping. This method overcomes limitations that prevent conventional point laser traps from effectively directing large particles. Expanding a previously developed line optical trap model into larger particle regimes, we verify and examine the advantages and limitations of diode laser bar trapping for manipulating particles greater than 100 µm in diameter within fluidic environments for biochemical, biological, and biomedical applications.

Thermal motion of a holographically trapped SPM-like probe

  • Stephen H Simpson and Simon Hanna

    By holding a complex object in multiple optical traps, it may be harmonically bound with respect to both its position and its orientation. In this way a small probe, or nanotool, can be manipulated in three dimensions and used to measure and apply directed forces, in the manner of a scanning probe microscope. In this paper we evaluate the thermal motion of such a probe held in holographic optical tweezers, by solving the Langevin equation for the general case of a set of spherical vertices linked by cylindrical rods. The concept of a corner frequency, familiar from the case of an optically trapped sphere, is appropriately extended to represent a set of characteristic frequencies given by the eigenvalues of the product of the stiffness matrix and the inverse hydrodynamic resistance matrix of the tool. These eigenvalues may alternatively be interpreted as inverses of a set of characteristic relaxation times for the system. The approach is illustrated by reference to a hypothetical tool consisting of a triangular arrangement of spheres with a lateral probe. The characteristic frequencies and theoretical resolution of the device are derived; variations of these quantities with tool size and orientation and with the optical power distribution, are also considered.

    Monday, September 14, 2009

    Combined optical trapping and microphotoluminescence of single InP nanowires

    Peter J. Reece, Suriati Paiman, Osama Abdul-Nabi, Qiang Gao, Michael Gal, H. Hoe Tan, and C. Jagadish

    In this letter, we demonstrate that microphotoluminescence may be combined with optical trapping for effective optical characterization of single target InP semiconductor nanowires in suspension. Using this technique, we may investigate structural properties of optically trapped nanowires, such as crystalline polytypes and stacking faults. This arrangement may also be used to resolve structural variations along the axis of the trapped nanowire.These results show that photoluminescence measurements may be coupled with optical tweezers without degrading the performance of the optical trap and provide a powerful interrogation tool for preselection of components for nanowire photonic devices.

    Using optical landscapes to control, direct and isolate aerosol particles

    Jon B. Wills, Jason R. Butler, John Palmer and Jonathan P. Reid

    We demonstrate the ability to direct the flow of aerosol droplets through a trapping cell using a tailored optical landscape generated by spatial light modulation. Using an optical barrier, droplets held in an optical trap can be effectively isolated from other droplets within the aerosol. To illustrate the effective isolation we compare the influence of different optical landscapes on the flow of free aerosol around a trapped droplet. We also present spectroscopic evidence of the optical barrier effect and apply the technique to permit controlled loading of different aerosol particles into neighbouring optical traps. This method will enable comparative measurements of aerosol properties to be made and facilitate the study of aerosol chemistry in sub-picolitre droplets. It also facilitates the use of an isolated droplet of known composition as a sensitive probe of the gas phase conditions in an aerosol ensemble.

    Optical tweezers with millikelvin precision of temperature-controlled objectives and base-pair resolution

    Mohammed Mahamdeh and Erik Schäffer

    In optical tweezers, thermal drift is detrimental for highr-esolution measurements. In particular, absorption of the trapping laser light by the microscope objective that focuses the beam leads to heating of the objective and subsequent drift. This entails long equilibration times which may limit sensitive biophysical assays. Here, we introduce an objective temperature feedback system for minimizing thermal drift. We measured that the infrared laser heated the objective by 0.7K per watt of laser power and that the laser focus moved relative to the sample by ≈1 nm/mK due to thermal expansion of the objective. The feedback stabilized the temperature of the trapping objective with millikelvin precision. This enhanced the long-term temperature stability and significantly reduced the settling time of the instrument to about 100 s after a temperature disturbance while preserving single DNA base-pair resolution of surface-coupled assays. Minimizing systematic temperature changes of the objective and concurrent drift is of interest for other high-resolution microscopy techniques. Furthermore, temperature control is often a desirable parameter in biophysical experiments.

    Calculating optical forces using the boundary integral method

    Per Jakobsen

    In this paper, we show that the boundary integral method is highly efficient for the calculation of optical forces on small dielectric and metallic objects. The boundary integral formulation for the Maxwell equations is stated, and an implementation of the equations is described, tested and used to derive new bistability results for two dielectric spheres in counterpropagating incoherent laser beams.

    Thursday, September 10, 2009

    Nondimensional analysis of particle behavior during cross-type optical particle separation

    Sang Bok Kim, Hyung Jin Sung, and Sang Soo Kim

    A nondimensional analysis of particle behavior during cross-type optical particle separation was performed. A new dimensionless number, S, was defined as the ratio of the optical force to the viscous drag force, and the effects of varying S on particle motion were examined. For large S, the particles undergo acceleration, deceleration, and release as they pass through the laser beam. The retention distance is much longer for large S than for small S. In addition, the effects on particle behavior of varying the wavelength of the laser beam, the particle size, and the index of refraction of the particles were investigated. Furthermore, an analytical expression of the retention distance for large S was validated.

    Direct observation of the binding state of the kinesin head to the microtubule

    Nicholas R. Guydosh & Steven M. Block

    The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules(1,2). Cargo attached to the kinesin stalk moves processively in 8-nm increments(3) as its twin motor domains (heads) carry out an asymmetric, 'hand-over-hand' walk(4-7). The extent of individual head interactions with the microtubule during stepping, however, remains controversial(4,8-14). A major experimental limitation has been the lack of a means to monitor the attachment of an individual head to the microtubule during movement, necessitating indirect approaches. Here we report the development of a single-molecule assay that can directly report head binding in a walking kinesin molecule, and show that only a single head is bound to the microtubule between steps at low ATP concentrations. A bead was linked to one of the two kinesin heads by means of a short DNA tether and used to apply rapidly alternating hindering and assisting loads with an optical trap. The time-dependent difference between forwards and backwards displacements of the bead alternated between two discrete values during stepping, corresponding to those intervals when the linked head adopted a bound or an unbound state. The linked head could only rebind the microtubule once ATP had become bound to its partner head.

    Effect of Energy Metabolism on Protein Motility in the Bacterial Outer Membrane

    Tabita Winther, Lei Xu, Kirstine Berg-Sorensen, Stanley Brown and Lene B. Oddershede

    We demonstrate the energy dependence of the motion of a porin, the lambda-receptor, in the outer membrane of living Escherichia coli by single molecule investigations. By poisoning the bacteria with arsenate and azide, the bacterial energy metabolism was stopped. The motility of individual lambda-receptors significantly and rapidly decreased upon energy depletion. We suggest two different causes for the ceased motility upon comprised energy metabolism: One possible cause is that the cell uses energy to actively wiggle its proteins, this energy being one order-of-magnitude larger than thermal energy. Another possible cause is an induced change in the connection between the lambda-receptor and the membrane structure, for instance by a stiffening of part of the membrane structure. Treatment of the cells with ampicillin, which directly targets the bacterial cell wall by inhibiting cross-linking of the peptidoglycan layer, had an effect similar to energy depletion and the motility of the lambda-receptor significantly decreased. Since the lambda-receptor is closely linked to the peptidoglycan layer, we propose that lambda-receptor motility is directly coupled to the constant and dynamic energy-consuming reconstruction of the peptidoglycan layer. The result of this motion could be to facilitate transport of maltose-dextrins through the porin.

    Friday, September 4, 2009

    Direct Measurement of the Nonconservative Force Field Generated by Optical Tweezers

    Pinyu Wu, Rongxin Huang, Christian Tischer, Alexandr Jonas, and Ernst-Ludwig Florin

    The force field of optical tweezers is commonly assumed to be conservative, neglecting the complex action of the scattering force. Using a novel method that extracts local forces from trajectories of an optically trapped particle, we measure the three-dimensional force fieldexperienced by a Rayleigh particle with 10 nm spatial resolution and femtonewton precision in force. We find that the force field is nonconservative with the nonconservative component increasing radially away from the optical axis, in agreement with the Gaussian beam model of the optical trap.

    Optical binding mechanisms: a conceptual model for Gaussian beam traps

    J. M. Taylor and G. D. Love

    Optical binding interactions between laser-trapped spherical microparticles are familiar in a wide range of trapping configurations. Recently it has been demonstrated that these experiments can be accurately modeled using Mie scattering or coupled dipole models. This can help confirm the physical phenomena underlying the inter-particle interactions, but does not necessarily develop a conceptual understanding of the effects that can lead to future predictions. Here we interpret results from a Mie scattering model to obtain a physical description which predict the behavior and trends for chains of trapped particles in Gaussian beam traps. In particular, it describes the non-uniform particle spacing and how it changes with the number of particles. We go further than simply demonstrating agreement, by showing that the mechanisms “hidden” within a mathematically and computationally demanding Mie scattering description can be explained in easily-understood terms.

    Photonic Binding in Silicon-Colloid Microcavities

    E. Xifré-Pérez, F. J. García de Abajo, R. Fenollosa, and F. Meseguer

    Photonic binding between two identical silicon-colloid-based microcavities is studied by using a generalized multipolar expansion. In contrast with previous works, we focus on low-order cavity modes that resemble low-energy electronic orbitals. For conservative light intensities, the interaction between cavity modes with moderate Q factors produces extremely large particle acceleration values. Optical forces dominate over van der Waals, gravity, and Brownian motion, and they show a binding-antibinding behavior in analogy to electronic binding. As these photonic forces are associated with relatively broad Mie mode resonances and they are not strongly influenced by sample absorption, our study opens a plausible avenue towards manipulation of high-refractive-index colloidal assemblies.

    The Q motif of a viral packaging motor governs its force generation and communicates ATP recognition to DNA interaction

    James M. Tsay, Jean Sippy, Michael Feiss and Douglas E. Smith

    A key step in the assembly of many viruses is the packaging of DNA into preformed procapsids by an ATP-powered molecular motor. To shed light on the motor mechanism we used single-molecule optical tweezers measurements to study the effect of mutations in the large terminase subunit in bacteriophage λ on packaging motor dynamics. A mutation, K84A, in the putative ATPase domain driving DNA translocation was found to decrease motor velocity by ≈40% but did not change the force dependence or decrease processivity substantially. These findings support the hypothesis that a deviant “Walker A-like” phosphate-binding motif lies adjacent to residue 84. Another mutation, Y46F, was also found to decrease motor velocity by ≈40% but also increase slipping during DNA translocation by >10-fold. These findings support the hypothesis that viral DNA packaging motors contain an adenine-binding motif that regulates ATP hydrolysis and substrate affinity analogous to the “Q motif” recently identified in DEAD-box RNA helicases. We also find impaired force generation for the Y46F mutant, which shows that the Q motif plays an important role in determining the power and efficiency of the packaging motor.
    A key step in the assembly of many viruses is the packaging of DNA into preformed procapsids by an ATP-powered molecular motor. To shed light on the motor mechanism we used single-molecule optical tweezers measurements to study the effect of mutations in the large terminase subunit in bacteriophage λ on packaging motor dynamics. A mutation, K84A, in the putative ATPase domain driving DNA translocation was found to decrease motor velocity by ≈40% but did not change the force dependence or decrease processivity substantially. These findings support the hypothesis that a deviant “Walker A-like” phosphate-binding motif lies adjacent to residue 84. Another mutation, Y46F, was also found to decrease motor velocity by ≈40% but also increase slipping during DNA translocation by >10-fold. These findings support the hypothesis that viral DNA packaging motors contain an adenine-binding motif that regulates ATP hydrolysis and substrate affinity analogous to the “Q motif” recently identified in DEAD-box RNA helicases. We also find impaired force generation for the Y46F mutant, which shows that the Q motif plays an important role in determining the power and efficiency of the packaging motor.

    Thursday, September 3, 2009

    ILPR G-Quadruplexes Formed in Seconds Demonstrate High Mechanical Stabilities

    Zhongbo Yu, Joseph D. Schonhoft, Soma Dhakal, Rabindra Bajracharya, Ravi Hegde, Soumitra Basuand Hanbin Mao

    The insulin linked polymorphism region (ILPR) is known to regulate transcription of the gene coding for insulin. The ILPR has guanine rich segments, suggesting that G quadruplexes may be responsible for this regulatory role. Using mechanical unfolding in a laser tweezers instrument and circular dichroism (CD) spectroscopy, we provide compelling evidence that highly stable parallel and antiparallel G quadruplex structures coexist in the predominant ILPR sequence of (ACAGGGGTGTGGGG)2 at a physiologically relevant concentration of 100 mM KCl. Experiments at the single molecular level have shown that unfolding forces for parallel and antiparallel structures (Funfold: 22.6 vs 36.9 pN, respectively) are higher than the stall forces of enzymes having helicase activities. From a mechanical perspective alone, these data support the hypothesis that G quadruplexes may cause replication slippage by blocking replication process. Using the unique combination of the rupture force and the contour length measured by laser tweezers, the simultaneous determination of probable parallel and antiparallel G quadruplex structures in a solution mixture has been achieved. Jarzynski′s equality analysis has revealed that the antiparallel G quadruplex is thermodynamically more stable than the parallel conformer (ΔG unfold: 23 vs 14 kcal/mol, respectively). On the other hand, kinetic measurements have indicated that both parallel and antiparallel structures fold rather rapidly (kfold: 0.4 vs 0.3 s−1, respectively), suggesting that they may be kinetically accessible for gene control. This work provides an unprecedented mechanical perspective on G quadruplex stability, presenting a unique opportunity to predict the functional consequence when motor enzymes encounter such structures.


    Rotation of birefringent particles in optical tweezers with spherical aberration

    Min-Cheng Zhong, Jin-Hua Zhou, Yu-Xuan Ren, Yin-Mei Li, and Zi-Qiang Wang

    Birefringent particles rotate when trapped in elliptically polarized light. When an infinity corrected oil-immersion objective is used for trapping, rotation of birefringent particles in optical tweezers based on an infinity optical microscope is affected by the spherical aberration at the glass-water interface. The maximum rotation rate of birefringent particles occurs close to the coverslip, and the rotation rate decreases dramatically as the trapped depth increases. We experimentally demonstrate that spherical aberration can be compensated by using a finite-distance-corrected objective to trap and rotate the birefringent particles. It is found that the trapped depth corresponding to the maximum rotation rate is 50 μm, and the rotation rates at deep trapped depths are improved.

    Raman Study of Mechanically Induced Oxygenation State Transition of Red Blood Cells Using Optical Tweezers

    Satish Rao,Štefan Bálint,Benjamin Cossins,Victor Guallar and Dmitri Petrov

    Raman spectroscopy was used to monitor changes in the oxygenation state of human red blood cells while they were placed under mechanical stress with the use of optical tweezers. The applied force is intended to simulate the stretching and compression that cells experience as they pass through vessels and smaller capillaries. In this work, spectroscopic evidence of a transition between the oxygenation and deoxygenation states, which is induced by stretching the cell with optical tweezers, is presented. The transition is due to enhanced hemoglobin-membrane and hemoglobin neighbor-neighbor interactions, and the latter was further studied by modeling the electrostatic binding of two of the protein structures.

    The Elastic Basis for the Shape of Borrelia burgdorferi

    Christopher Dombrowski, Wanxi Kan, Md. Abdul Motaleb, Nyles W. Charon, Raymond E. Goldstein and Charles W. Wolgemuth

    The mechanisms that determine bacterial shape are in many ways poorly understood. A prime example is the Lyme disease spirochete, Borrelia burgdorferi (B. burgdorferi), which mechanically couples its motility organelles, helical flagella, to its rod-shaped cell body, producing a striking flat-wave morphology. A mathematical model is developed here that accounts for the elastic coupling of the flagella to the cell cylinder and shows that the flat-wave morphology is in fact a natural consequence of the geometrical and material properties of the components. Observations of purified periplasmic flagella show two flagellar conformations. The mathematical model suggests that the larger waveform flagellum is the more relevant for determining the shape of B. burgdorferi. Optical trapping experiments were used to measure directly the mechanical properties of these spirochetes. These results imply relative stiffnesses of the two components, which confirm the predictions of the model and show that the morphology of B. burgdorferi is completely determined by the elastic properties of the flagella and cell body. This approach is applicable to a variety of other structures in which the shape of the composite system is markedly different from that of the individual components, such as coiled-coil domains in proteins and the eukaryotic axoneme.

    Marker-free cell discrimination by holographic optical tweezers

    F. Schaal, M. Warber, S. Zwick, H. van der Kuip, T. Haist, W. Osten

    We introduce a method for marker-free cell discrimination based on optical tweezers. Cancerous, non-cancerous, and drug-treated cells could be distinguished by measuring the trapping forces using holographic optical tweezers. We present trapping force measurements on different cell lines: normal pre-B lymphocyte cells (BaF3; "normal cells"), their Bcr-Abl transformed counterparts (BaF3-p185; "cancer cells") as a model for chronic myeloid leukaemia (CML) and Imatinib treated BaF3-p185 cells. The results are compared with reference measurements obtained by a commercial flow cytometry system.