Force generation in small ensembles of Brownian motors

The motility of certain gram-negative bacteria is mediated by retraction of type IV pili surface filaments, which are essential for infectivity. The retraction is powered by a strong molecular motor protein, PilT, producing very high forces that can exceed 150 pN. The molecular details of the motor mechanism are still largely unknown, while other features have been identified, such as the ring-shaped protein structure of the PilT motor. The surprisingly high forces generated by the PilT system motivate a model investigation of the generation of large forces in molecular motors. We propose a simple model, involving a small ensemble of motor subunits interacting through the deformations on a circular backbone with finite stiffness. The model describes the motor subunits in terms of diffusing particles in an asymmetric, time-dependent binding potential (flashing ratchet potential), roughly corresponding to the ATP hydrolysis cycle. We compute force-velocity relations in a subset of the parameter space and explore how the maximum force (stall force) is determined by stiffness, binding strength, ensemble size, and degree of asymmetry. We identify two qualitatively different regimes of operation depending on the relation between ensemble size and asymmetry. In the transition between these two regimes, the stall force depends nonlinearly on the number of motor subunits. Compared to its constituents without interactions, we find higher efficiency and qualitatively different force-velocity relations. The model captures several of the qualitative features obtained in experiments on pilus retraction forces, such as roughly constant velocity at low applied forces and insensitivity in the stall force to changes in the ATP concentration.Comment: RevTex 9 pages, 4 figures. Revised version, new subsections in Sec. III, removed typo

Liquid-Jet-Target Microfocus X-Ray Sources : Electron Guns, Optics and Phase-Contrast Imaging

This Thesis describes the development of an electron-impact microfocus x-ray source and its application for phase-contrast imaging. The source is based on a novel, liquid-jet target concept. Stable and continuous operation can be achieved at substantially higher electron-beam power densities than conventional solid target based systems. The maximum x-ray brightness can potentially be increased by a factor of 10-1000, which would provide significantly improved performance in applications such as imaging. In order to reach the high x-ray brightness, comparable performance from the electron gun is needed. A LaB6-cathode-based electron gun is analyzed in terms of achievable e-beam brightness and beam quality and is found capable to deliver power densities in the 10-100 MW/mm2 range using optimized electro-optics. A proof-of-principle microfocus source has been developed. Experiments show that the liquid-metal-jet target can be operated at more than an order of magnitude higher e-beam power densities than modern solid-metal targets. This brightness enhancement has been utilized to acquire in-line phase-contrast images of weakly absorbing objects. The source potentially enables the application of high-resolution phase-contrast x-ray imaging with short exposure times in clinics and laboratories. Different liquid-jet-target materials have been tested. The Sn-jet (Ka=25.3 keV) could be suitable for mammography, whereas the Ga-jet ((Ka=9.2 keV) may be utilized for x-ray diffraction studies. In addition, a non-metallic methanol jet has been the demonstrated in stable x-ray operation. All materials and compounds found in liquid form can, thus, potentially be used for electron-impact liquid-jet-target x-ray generation. Scaling to higher e-beam power density and x-ray brightness levels is discussed and is determined to be feasible. Potential difficulties, such as debris emission and instabilities of the x-ray emission spot, are investigated in some detail. Larger and/or faster jets could overcome the present limitations because of their inherently higher heat load capacities. Dynamic-similarity experiments show that liquid jets can in principle be operated in a stable manner at much higher speeds than previously shown.QC 2010091

Liquid-Jet-Target Microfocus X-Ray Sources : Electron Guns, Optics and Phase-Contrast Imaging

This Thesis describes the development of an electron-impact microfocus x-ray source and its application for phase-contrast imaging. The source is based on a novel, liquid-jet target concept. Stable and continuous operation can be achieved at substantially higher electron-beam power densities than conventional solid target based systems. The maximum x-ray brightness can potentially be increased by a factor of 10-1000, which would provide significantly improved performance in applications such as imaging. In order to reach the high x-ray brightness, comparable performance from the electron gun is needed. A LaB6-cathode-based electron gun is analyzed in terms of achievable e-beam brightness and beam quality and is found capable to deliver power densities in the 10-100 MW/mm2 range using optimized electro-optics. A proof-of-principle microfocus source has been developed. Experiments show that the liquid-metal-jet target can be operated at more than an order of magnitude higher e-beam power densities than modern solid-metal targets. This brightness enhancement has been utilized to acquire in-line phase-contrast images of weakly absorbing objects. The source potentially enables the application of high-resolution phase-contrast x-ray imaging with short exposure times in clinics and laboratories. Different liquid-jet-target materials have been tested. The Sn-jet (Ka=25.3 keV) could be suitable for mammography, whereas the Ga-jet ((Ka=9.2 keV) may be utilized for x-ray diffraction studies. In addition, a non-metallic methanol jet has been the demonstrated in stable x-ray operation. All materials and compounds found in liquid form can, thus, potentially be used for electron-impact liquid-jet-target x-ray generation. Scaling to higher e-beam power density and x-ray brightness levels is discussed and is determined to be feasible. Potential difficulties, such as debris emission and instabilities of the x-ray emission spot, are investigated in some detail. Larger and/or faster jets could overcome the present limitations because of their inherently higher heat load capacities. Dynamic-similarity experiments show that liquid jets can in principle be operated in a stable manner at much higher speeds than previously shown.QC 2010091

Structural characterization of core-bradavidin in complex with biotin

<div><p>Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria <i>Bradyrhizobium diazoefficiens</i>. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (<i>i</i>.<i>e</i>. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from <i>Rhodopseudomonas palustris</i>, and of an avidin-like protein from <i>Bradyrhizobium sp</i>. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in <i>E</i>. <i>coli</i>, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.</p></div