High-resolution imaging of 2D outer membrane protein F (OmpF) crystals by atomic force microscopy

In this chapter the methodological bases are provided to achieve subnanometer resolution on two-dimensional (2D) membrane protein crystals by atomic force microscopy (AFM). This is outlined in detail with the example of AFM studies of the outer membrane protein F (OmpF) from the bacterium Escherichia coli (E. coli). We describe in detail the high-resolution imaging of 2D OmpF crystals in aqueous solution and under near-physiological conditions. The topographs of OmpF, and stylus effects and artifacts encountered when imaging by AFM are discussed.

Cooperative and Noncooperative Assembly of Oligopyrenotides Resolved by Atomic Force Microscopy

The supramolecular assembly of amphiphilic oligopyrenotide building blocks (covalently linked heptapyrene, Py7) is studied by atomic force microscopy (AFM) in combination with optical spectroscopy. The assembly process is triggered in a controlled manner by increasing the ionic strength of the aqueous oligomer solution. Cooperative noncovalent interactions between individual oligomeric units lead to the formation of DNA-like supramolecular polymers. We also show that the terminal attachment of a single cytidine nucleotide to the heptapyrenotide (Py7-C) changes the association process from a cooperative (nucleation−elongation) to a noncooperative (isodesmic) regime, suggesting a structure misfit between the cytidine and the pyrene units. We also demonstrate that AFM enables the identification and characterization of minute concentrations of the supramolecular products, which was not accessible by conventional optical spectroscopy.

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Atomic force microscopy for the study of membrane proteins

Fundamental biological processes such as cell-cell communication, signal transduction, molecular transport and energy conversion are performed by membrane proteins. These important proteins are studied best in their native environment, the lipid bilayer. The atomic force microscope (AFM) is the instrument of choice to determine the native surface structure, supramolecular organization, conformational changes and dynamics of membrane-embedded proteins under near-physiological conditions. In addition, membrane proteins are imaged at subnanometer resolution and at the single molecule level with the AFM. This review highlights the major advances and results achieved on reconstituted membrane proteins and native membranes as well as the recent developments of the AFM for imaging.

Morphology Change of C60 Islands on Organic Crystals Observed by Atomic Force Microscopy

Organic-organic heterojunctions are nowadays highly regarded materials for light-emitting diodes, field-effect transistors, and photovoltaic cells with the prospect of designing low-cost, flexible, and efficient electronic devices.1-3 However, the key parameter of optimized heterojunctions relies on the choice of the molecular compounds as well as on the morphology of the organic-organic interface,4 which thus requires fundamental studies. In this work, we investigated the deposition of C60 molecules at room temperature on an organic layer compound, the salt bis(benzylammonium)bis(oxalato)cupurate(II), by means of noncontact atomic force microscopy. Three-dimensional molecular islands of C60 having either triangular or hexagonal shapes are formed on the substrate following a "Volmer-Weber" type of growth. We demonstrate the dynamical reshaping of those C60 nanostructures under the local action of the AFM tip at room temperature. The dissipated energy is about 75 meV and can be interpreted as the activation energy required for this migration process.

Electrochemical current-sensing atomic force microscopy in conductive solutions

Insulated atomic force microscopy probes carrying gold conductive tips were fabricated and employed as bifunctional force and current sensors in electrolyte solutions under electrochemical potential control. The application of the probes for current-sensing imaging, force and current–distance spectroscopy as well as scanning electrochemical microscopy experiments was demonstrated.

Fabrication of cone-shaped boron doped diamond and gold nanoelectrodes for AFM-SECM

We demonstrate a reliable microfabrication process for a combined atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) measurement tool. Integrated cone-shaped sensors with boron doped diamond (BDD) or gold (Au) electrodes were fabricated from commercially available AFM probes. The sensor formation process is based on mature semiconductor processing techniques, including focused ion beam (FIB) machining, and highly selective reactive ion etching (RIE). The fabrication approach preserves the geometry of the original AFM tips resulting in well reproducible nanoscaled sensors. The feasibility and functionality of the fully featured tips are demonstrated by cyclic voltammetry, showing good agreement between the measured and calculated currents of the cone-shaped AFM-SECM electrodes.

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F(1)F(o)-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F(1)F(o)-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F(1)F(o)-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F(1)F(o)-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F(1) catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F(1) to the lipid monolayer and the F(o) membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F(o) by electron microscopy and AFM.

Formation of supramolecular polymers by chrysene oligomers

Supramolecular assembly of π-conjugated systems is of large interested due to the possibility to use them in electronic devices.[1] Chrysene is a polyaromatic hydrocarbon which has been studied e.g for organic light-emitting diodes (OLEDs).[2] In continuation of our previous work involving the supramolecular polymerisation of pyrene oligomers [3] an oligomer consisting of three chrysenes linked by phophodiesters was synthesised (Chry3). UV-Vis measurements show that aggregates of Chry3 are formed in aqueous medium. This is illustrated by general hypochromicity, a change in vibronic band intensities and, in particular, the appearance of a red-shifted absorption band in the S0 → S2 transition. The data suggest the formation of J-aggregates. The formation of supramolecular polymers is further studied by temperature-dependent absorption- and fluorescence measurements, and by atomic force microscopy (AFM).

Measuring localized viscoelasticity of the vitreous body using intraocular microprobes.

Vitrectomy is a standard ophthalmic procedure to remove the vitreous body from the eye. The biomechanics of the vitreous affects its duration (by changing the removal rate) and the mechanical forces transmitted via the vitreous on the surrounding tissues during the procedure. Biomechanical characterization of the vitreous is essential for optimizing the design and control of instruments that operate within the vitreous for improved precision, safety, and efficacy. The measurements are carried out using a magnetic microprobe inserted into the vitreous, a method known as magnetic microrheology. The location of the probe is tracked by a microscope/camera while magnetic forces are exerted wirelessly by applied magnetic fields. In this work, in vitro artificial vitreous, ex vivo human vitreous and ex vivo porcine vitreous were characterized. In addition, in vivo rabbit measurements were performed using a suturelessly injected probe. Measurements indicate that viscoelasticity parameters of the ex vivo human vitreous are an order of magnitude different from those of the ex vivo porcine vitreous. The in vivo intra-operative measurements show typical viscoelastic behavior of the vitreous with a lower compliance than the ex vivo measurements. The results of the magnetic microrheology measurements were validated with those obtained by a standard atomic force microscopy (AFM) method and in vitro artificial vitreous. This method allows minimally-invasive characterization of localized mechanical properties of the vitreous in vitro, ex vivo, and in vivo. A better understanding of the characteristics of the vitreous can lead to improvements in treatments concerning vitreal manipulation such as vitrectomy.

Structure determination of channel and transport proteins by high-resolution microscopy techniques

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.

Immobilization and characterization of recombinant esterase enzyme on chitosan nanoparticles

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres’ average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60°C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).