Chandra S. Ramanujan

Direct measurement of molecular stiffness and damping in confined water layers

We present direct and linear measurements of the normal stiffness and damping of a confined, few molecule thick water layer. The measurements were obtained by use of a small amplitude

Electrically conducting, ultra-sharp, high aspect-ratio probes for AFM fabricated by electron-beam-induced deposition of platinum.

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AFM observation of single, functioning ionotropic glutamate receptors reconstituted in lipid bilayers

, off-resonance atomic force microscopy technique. We measured stiffness and damping oscillations revealing up to seven molecular layers separated by

Self-assembly of vesicle nanoarrays on Si: A potential route to high-density functional protein arrays

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Reconstitution of homomeric GluA2flop receptors in supported lipid membranes: functional and structural properties

. Relaxation times could also be calculated and were found to indicate a significant slow-down of the dynamics of the system as the confining separation was reduced. We found that the dynamics of the system is determined not only by the interfacial pressure, but more significantly by solvation effects which depend on the exact separation of tip and surface. The dynamic forces reflect the layering of the water molecules close to the mica surface and are enhanced when the tip-surface spacing is equivalent to an integer multiple of the size of the water molecules. We were able to model these results by starting from the simple assumption that the relaxation time depends linearly on the film stiffness.

Bionanotechnology with Membrane Proteins: Mechanics and Electronics

We report on the fabrication of electrically conducting, ultra-sharp, high-aspect ratio probes for atomic force microscopy by electron-beam-induced deposition of platinum. Probes of 4.0 ±1.0 nm radius-of-curvature are routinely produced with high repeatability and near-100% yield. Contact-mode topographical imaging of the granular nature of a sputtered gold surface is used to assess the imaging performance of the probes, and the derived power spectral density plots are used to quantify the enhanced sensitivity as a function of spatial frequency. The ability of the probes to reproduce high aspect-ratio features is illustrated by imaging a close-packed array of nanospheres. The electrical resistance of the probes is measured to be of order 100 kΩ.

Elastic modulus of suspended purple membrane measured by atomic force microscopy

BACKGROUND Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment. METHODS Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate. RESULTS Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements. CONCLUSIONS Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment. GENERAL SIGNIFICANCE AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications.

STAGE FOR SCANNING PROBE MICROSCOPY AND SAMPLE OBSERVATION METHOD

The authors show that 100nm unilamellar thiol-tagged vesicles bind discretely and specifically to Au nanodots formed on a Si surface. An array of such dots, consisting of 20nm Au–Si three-dimensional islands, is formed by self-assembly on terraces of small-angle-miscut Si(111) after Au deposition. Consequently, both the formation of the nanopattern and the subsequent attachment of the vesicles are self-organized and occur without the need for any “top-down” lithographic processes. This approach has the potential to provide the basis of a low-cost, high-density nanoarray for use in proteomics and drug discovery.

Liposome, Proteoliposome, Biochip, and Method for Producing Liposome and Proteoliposome

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2(flop) receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.Background: Ionotropic glutamate receptors mediate fast excitatory synaptic transmission in the vertebrate CNS. Results: Conformational flexibility and dimensions of functional membrane-embedded full-length GluA2flop receptors are characterized by atomic force microscopy. Conclusion: Conformational flexibility and dimensions are both strongly affected by receptor density. Significance: The reconstitution protocol lays the foundation for correlated structure-function analysis of membrane-embedded glutamate receptors. AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.

Reconstitution of Homomeric GluA2 flop Receptors in Supported Lipid Membranes FUNCTIONALANDSTRUCTURALPROPERTIES

Bionanotechnoloy IRC, Physics Department, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU, Oxfordshire, United Kingdom Phone: +44-1865-272269 E-mail: s.antoranzcontera1@physics.ox.ac.uk 2 Biochemistry Department, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom Molecular and Bio-Science Research Group, NTT Basic Research Laboratories, NTT Corporation, 3-1 MorinosatoWakamiya, Atsugi, Kanagawa 243-0198, Japan