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.
Thursday, September 10, 2009
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.
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.
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.
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.
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.
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
DOI
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.
DOI
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.
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.
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.
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.
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.
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