Characterization of single-molecule pentanedithiol junctions by inelastic electron tunneling spectroscopy and first-principles calculations

We study pentanedithiol molecular junctions formed by means of the break-junction technique with a scanning tunneling microscope at low temperatures. Using inelastic electron tunneling spectroscopy and first-principles calculations, the response of the junction to elastic deformation is examined. We show that this procedure makes a detailed characterization of the molecular junction possible. In particular, our results indicate that tunneling takes place through just a single molecule.Comment: 5 pages, 4 figures (accepted in Phys. Rev. B

Microfilaments and actin-associated proteins at sites of membrane-substrate attachment within acetylcholine receptor clusters

Rat myotubes in tissue culture form broad areas of close contact with the substrate. These areas often display two distinct, interdigitating sets of membrane domains. One, the "contact domain", is close to the substrate; the other, termed the "AChR domain", is further from the substrate and is rich in acetylcholine receptors (AChR). We have used fluorescence techniques to study the organization of the cytoskeleton in these areas. Substrate-apposed membrane of the myotubes was exposed either by shearing or by permeabilizing the cells with a neutral detergent. Phalloidin derivatives and affinity-purified polyclonal or monoclonal antibodies specific for cytoskeletal proteins were then applied to the samples. Sheared samples were observed by epifluorescence microscopy; detergent-permeabilized samples were observed by total internal reflection fluorescence microscopy. We found that, like antivinculin, fluorescent phalloidin derivatives and antibodies to [alpha]-actinin, filamin, and talin preferentially labeled the contact domains. This suggests that bundles of microfilaments associate with the membrane at sites of myotubesubstrate attachment. In contrast, a 43K protein, closely associated with AChR, was present only at AChR domains. A monoclonal antibody to actin labeled both AChR and contact domains, suggesting that actin is enriched over both regions. Our results suggest that, like the plasma membrane of AChR clusters, the underlying membrane skeleton is organized into at least two distinct domains.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27926/1/0000350.pd

Techniques to Analyze sRNA Protein Cofactor Self-Assembly In Vitro

Post-transcriptional control of gene expression by small regulatory noncoding RNA (sRNA) needs protein accomplices to occur. Past research mainly focused on the RNA chaperone Hfq as cofactor. Nevertheless, recent studies indicated that other proteins might be involved in sRNA-based regulations. As some of these proteins have been shown to self-assemble, we describe in this chapter protocols to analyze the nano-assemblies formed. Precisely, we focus our analysis on Escherichia coli Hfq as a model, but the protocols presented here can be applied to analyze any polymer of proteins. This chapter thus provides a guideline to develop commonly used approaches to detect prokaryotic protein self-assembly, with a special focus on the detection of amyloidogenic polymers

Fluorescent studies of acetylcholine receptor clustering.

We have used various novel fluorescent microscopy techniques to study the clustering mechanism of the acetylcholine receptor (AChR) in primary cultures of rat myotubes. First, we further developed a technique, Polarized Fluorescence Photobleaching Recovery (PFPR), for measuring rotational diffusion in solutions and membranes. The technique is applicable to a wide range of time scales, from microseconds to seconds. Furthermore, it does not require deoxygenation, thereby making it suitable for the study of protein dynamics on living membranes. The technique was experimentally verified on small latex beads with a variety of diameters, common fluorophore labels, and solvent viscosities. We then measured the rotational motion of the AChR (around an axis perpendicular to the plane of the membrane) in various aggregational states on the surface of living rat myotubes. We found that nonclustered receptors are an heterogeneous mixture of several rotating components: a very fast component (D\sb{\rm r} > 5000 s\sp{-1}, $\cong$31%), two intermediate components (D\sb{\rm r} \cong 700 $\pm$ 80 s\sp{-1}, \cong15%, and D\sb{\rm r} \cong 2.9 $\pm$.6 s\sp{-1}, \cong30%) and an immobile component (D\sb{\rm r} < 0.1 s\sp{-1}, \cong24%). Clustered receptors, as well as clusters treated with carbachol, a receptor agonist that has a cluster disaggregating effect, show no rotation up to 300 ms (D\sb{\rm r} < 0.1 s\sp{-1}). Another fluorescent technique used, Total Internal Reflection Fluorescence (TIRF) allowed us to look selectively at cytoskeletal proteins that codistribute with the cluster receptors. We found that: (a) the 43K protein codistributes with the clustered receptor molecules; (b) $\alpha$-actinin, filamin and vinculin are found associated preferentially with the receptor-poor regions of the cluster; (c) actin is found in both, receptor-rich and receptor poor regions of clusters. Scanning Fluorescence Correlation Spectroscopy (S-FCS) was used to measure the number of receptors in the microclusters constituting the large endogenous clusters present in the cultured myotubes. The values measured ranged from 200 to 13000 receptors per microcluster. This large range of values is due to biological variability between clusters. In summary: the technique of PFPR can be used to measure molecular rotational diffusion rates; different states of aggregation of the AChRs in the surface of cultured rat myotubes can be correlated with different rotational behavior of the receptors; nonclustered receptors are free to rotate whereas clustered ones are rotationally immobile; and clustered receptors present a possible close association with actin and 43K protein and no direct association with vinculin, $\alpha$-actinin and filamin.Ph.D.Biological SciencesBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128395/2/9001730.pd

CdSe/CdS nanoparticles immobilized on pNIPAm-based microspheres

In this work, CdSe/CdS semiconductor ligand-exchanged nanoparticles have been immobilized on poly-(N-isopropylacrylamide) (pNIPAM)-based microspheres. The size and the shrinkage capacity of the spheres can be tuned by the ratio of NIPAM/styrene (pNIPAM-PS spheres) or NIPAM/BIS (N-N'-methylene-bis-acrylamide) and MA (maleic acid) (pNIPAM-BIS-MA spheres). A ligand-exchange procedure for the transfer of initially organic compatible nanoparticles into aqueous solution using amine-modified or thiol-modified poly(ethylene oxide)s (PEOs) has been carried out prior to their immobilization. We observed that the interaction of the nanoparticles with the pNIPAM-based system depends on the nature of the ligands and the chemical composition of the microspheres. Nanoparticles capped with amine-or mercapto-poly(ethylene oxide) s ligands interact with pNIPAM-PS beads while only amine-capped ones show a clear tendency to interact with pNIPAM containing acid groups which leads to a high nanoparticle coverage. Dynamic light scattering measurements, atomic force microscopy and optical spectroscopy hint that nanoparticles are placed on the surface of pNIPAM-BIS-MA beads while being partially incorporated into pNIPAM-PS network. Cell culture studies demonstrate that the fluorescent composites show non-specific binding to fibroblasts. These features may be very valuable to develop materials for drug delivery and specific targeting of cells combined with the outstanding optical properties of semiconductor nanoparticles as fluorescent labelers

Useful Oriented Immobilization of Antibodies on Chimeric Magnetic Particles: Direct Correlation of Biomacromolecule Orientation with Biological Activity by AFM Studies

The preparation and performance of a suitable chimeric biosensor basedon antibodies (Abs) immobilized on lipase-coated magnetic particles by means of astanding orienting strategy are presented. This novel system is based on hydrophobicmagnetic particles coated with modified lipase molecules able to orient and furtherimmobilize different Abs in a covalent way without any previous site-selective chemicalmodification of biomacromolecules. Different key parameters attending the process werestudied and optimized. The optimal preparation was performed using a controlled loading(1 nmol Ab g−1chimeric support) at pH 9 and a short reaction time to recover abiological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and thefinal achievement of ahighly active and recyclable chimeric immune sensor. This direct technique wasdemonstrated to be a powerful alternative to the indirect immunoactivity assay methodsfor the study of biomacromolecule-oriented immobilizations.Centro Superior de Investigaciones CientíficasEuropean CommissionDepto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

Extracting the Shape and Size of Biomolecules Attached to a Surface as Suspended Discrete Nanoparticles

The ability to derive information on the conformation of surface attached biomolecules by using simple techniques such as biosensors is currently considered of great importance in the fields of surface science and nanotechnology. Here we present a nanoshape sensitive biosensor where a simple mathematical expression is used to relate acoustic measurements to the geometrical features of a surface-attached biomolecule. The underlying scientific principle is that the acoustic ratio (Δ<i>D</i>/Δ<i>F</i>) is a measure of the hydrodynamic volume of the attached entity, mathematically expressed by its intrinsic viscosity [η]. A methodology is presented in order to produce surfaces with discretely bound biomolecules where their native conformation is maintained. Using DNA anchors we attached a spherical protein (streptavidin) and a rod-shaped DNA (47bp) to a quartz crystal microbalance (QCM) device in a suspended way and predicted correctly through acoustic measurements their conformation, i.e., shape and length. The methodology can be widely applied to draw conclusions on the conformation of any biomolecule or nanoentity upon specific binding on the surface of an acoustic wave device

Publisher Correction: Membrane association of the bacterial riboregulator Hfq and functional perspectives

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper

Membrane association of the bacterial riboregulator Hfq and functional perspectives

Abstract Hfq is a bacterial RNA binding protein that carries out several roles in genetic expression regulation, mainly at the post-transcriptional level. Previous studies have shown its importance in growth and virulence of bacteria. Here, we provide the direct observation of its ability to interact with membranes. This was established by co-sedimentation assay, cryo-transmission electron (cryo-TEM) and atomic force (AFM) microscopies. Furthermore, our results suggest a role for its C-terminus amyloidogenic domain in membrane disruption. Precisely, AFM images of lipid bilayers in contact with Hfq C-terminus fibrils show the emergence of holes with a size dependent on the time of interaction. Cryo-TEM observations also show that liposomes are in contact with clusters of fibrils, with occasional deformation of the vesicles and afterward the apparition of a multitude of tiny vesicles in the proximity of the fibrils, suggesting peptide-induced breakage of the liposomes. Finally, circular dichroism spectroscopy demonstrated a change in the secondary structure of Hfq C-terminus upon interaction with liposomes. Altogether, these results show an unexpected property of Hfq and suggest a possible new role for the protein, exporting sRNA outside of the bacterial cell