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.

Gi regulation of secretory vesicle swelling examined by atomic force microscopy

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the α subunit of a heterotrimeric GTP binding protein Gαi3 and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15–25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that Gαi3 protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy.

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with the ROMK1-GST fusion protein's existence as a heterodimer. In experiments on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solution produced a change in height of the aggregates. This change (which was reversible) suggests that ATP induces a structural change in the ROMK1 protein. The data show that atomic force microscopy is a useful tool for examination of purified protein molecules under near-physiological conditions, and furthermore, that structural alterations in the proteins may be continuously investigated.

Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy.

We have used self-assembled purines and pyrimidines on planar gold surfaces and on gold-coated atomic force microscope (AFM) tips to directly probe intermolecular hydrogen bonds. Electron spectroscopy for chemical analysis (ESCA) and thermal programmed desorption (TPD) measurements of the molecular layers suggested monolayer coverage and a desorption energy of about 25 kcal/mol. Experiments were performed under water, with all four DNA bases immobilized on AFM tips and flat surfaces. Directional hydrogen-bonding interaction between the tip molecules and the surface molecules could be measured only when opposite base-pair coatings were used. The directional interactions were inhibited by excess nucleotide base in solution. Nondirectional van der Waals forces were present in all other cases. Forces as low as two interacting base pairs have been measured. With coated AFM tips, surface chemistry-sensitive recognition atomic force microscopy can be performed.

Nanomechanical characterization of soft bioelectronic interfaces via modeling-informed atomic force microscopy

The field of bioelectronics involves the use of electrodes to exchange electrical signals with biological systems for diagnostic and therapeutic purposes in biomedical devices and healthcare applications. However, the mechanical compatibility of implantable devices with the human body has been a challenge, particularly with long-term implantation into target organs. Current rigid bioelectronics can trigger inflammatory responses and cause unstable device functions due to the mechanical mismatch with the surrounding soft tissue. Recent advances in flexible and stretchable electronics have shown promise in making bioelectronic interfaces more biocompatible. To fully achieve this goal, material science and engineering of soft electronic devices must be combined with quantitative characterization and modeling tools to understand the mechanical issues at the interface between electronic technology and biological tissue. Local mechanical characterization is crucial to understand the activation of failure mechanisms and optimizing the devices. Experimental techniques for testing mechanical properties at the nanoscale are emerging, and the Atomic Force Microscope (AFM) is a good candidate for in situ local mechanical characterization of soft bioelectronic interfaces. In this work, in situ experimental techniques with solely AFM supported by interpretive models for the characterization of planar and three-dimensional devices suitable for in vivo and in vitro biomedical experimentations are reported. The combination of the proposed models and experimental techniques provides access to the local mechanical properties of soft bioelectronic interfaces. The study investigates the nanomechanics of hard thin gold films on soft polymeric substrates (Poly(dimethylsiloxane) PDMS) and 3D inkjet-printed micropillars under different deformation states. The proposed characterization methods provide a rapid and precise determination of mechanical properties, thus giving the possibility to parametrize the microfabrication steps and investigate their impact on the final device.

Coerced Mechanical Coarsening of Nanoparticle Assemblies

Coarsening is a ubiquitous phenomenon [1-3] that underpins countless processes in nature, including epitaxial growth [1,3,4], the phase separation of alloys, polymers and binary fluids [2], the growth of bubbles in foams5, and pattern formation in biomembranes6. Here we show, in the first real-time experimental study of the evolution of an adsorbed colloidal nanoparticle array, that tapping-mode atomic force microscopy (TM-AFM) can drive the coarsening of Au nanoparticle assemblies on silicon surfaces. Although the growth exponent has a strong dependence on the initial sample morphology, our observations are largely consistent with modified Ostwald ripening processes [7-9]. To date, ripening processes have been exclusively considered to be thermally activated, but we show that nanoparticle assemblies can be mechanically coerced towards equilibrium, representing a new approach to directed coarsening. This strategy enables precise control over the evolution of micro- and nanostructures.

Large fluctuations in the disassembly rate of microtubules revealed by atomic force microscopy.

Atomic force microscopy (AFM) in situ has been used to observe the cold disassembly dynamics of microtubules at a previously unrealised spatial resolution. Microtubules either electrostatically or covalently bound to aminosilane surfaces disassembled at room temperature under buffer solutions with no free tubulin present. This process was followed by taking sequential tapping-mode AFM images and measuring the change in the microtubule end position as a function of time, with an spatial accuracy down to +/-20nm and a temporal accuracy of +/-1s. As well as giving average disassembly rates on the order of 1-10 tubulin monomers per second, large fluctuations in the disassembly rate were revealed, indicating that the process is far from smooth and linear under these experimental conditions. The surface bound rates measured here are comparable to the rates for GMPCPP-tubulin microtubules free in solution, suggesting that inhibition of tubulin curvature through steric hindrance controls the average, relatively low disassembly rate. The large fluctuations in this rate are thought to be due to multiple pathways in the kinetics of disassembly with differing rate constants and/or stalling due to defects in the microtubule lattice. Microtubules that were covalently bound to the surface left behind the protofilaments covalently cross-linked to the aminosilane via glutaraldehyde during the disassembly process. Further work is needed to quantitatively assess the effects of surface binding on protofibril disassembly rates, reveal any differences in disassembly rates between the plus and minus ends and to enable assembly as well as disassembly to be imaged in the microscope fluid cell in real-time.

Relationship Between Surface Topography and Energy Density Distribution of Er,Cr:YSGG Beam on Irradiated Dentin: An Atomic Force Microscopy Study

Objective: The aim of this study was to assess by atomic force microscopy (AFM) the effect of Er,Cr:YSGG laser application on the surface microtopography of radicular dentin. Background: Lasers have been used for various purposes in dentistry, where they are clinically effective when used in an appropriate manner. The Er, Cr: YSGG laser can be used for caries prevention when settings are below the ablation threshold. Materials and Methods: Four specimens of bovine dentin were irradiated using an Er, Cr:YSGG laser (lambda = 2.78 mu m), at a repetition rate of 20 Hz, with a 750-mu m-diameter sapphire tip and energy density of 2.8 J/cm(2) (12.5 mJ/pulse). After irradiation, surface topography was analyzed by AFM using a Si probe in tapping mode. Quantitative and qualitative information concerning the arithmetic average roughness (Ra) and power spectral density analyses were obtained from central, intermediate, and peripheral areas of laser pulses and compared with data from nonirradiated samples. Results: Dentin Ra for different areas were as follows: central, 261.26 (+/- 21.65) nm; intermediate, 83.48 (+/- 6.34) nm; peripheral, 45.8 (+/- 13.47) nm; and nonirradiated, 35.18 (+/- 2.9) nm. The central region of laser pulses presented higher ablation of intertubular dentin, with about 340-760 nm height, while intermediate, peripheral, and nonirradiated regions presented no difference in height of peritubular and interperitubular dentin. Conclusion: According to these results, we can assume that even when used at a low-energy density parameter, Er, Cr: YSGG laser can significantly alter the microtopography of radicular dentin, which is an important characteristic to be considered when laser is used for clinical applications.

Young’s modulus of silicon nitride used in scanning force microscope cantilevers

The Young’s modulus and Poisson’s ratio of high-quality silicon nitride films with 800 nm thickness, grown on silicon substrates by low-pressure chemical vapor deposition, were determined by measuring the dispersion of laser-induced surface acoustic waves. The Young’s modulus was also measured by mechanical tuning of commercially available silicon nitride cantilevers, manufactured from the same material, using the tapping mode of a scanning force microscope. For this experiment, an expression for the oscillation frequencies of two-media beam systems is derived. Both methods yield a Young’s modulus of 280–290 GPa for amorphous silicon nitride, which is substantially higher than previously reported (E5146 GPa). For Poisson’s ratio, a value of n 50.20 was obtained. These values are relevant for the determination of the spring constant of the cantilever and the effective tip–sample stiffness

DNA fragmentation by gamma radiation and electron beams using atomic force microscopy

Double-stranded pBS plasmid DNA was irradiated with gamma rays at doses ranging from 1 to 12 kGy and electron beams from 1 to 10 kGy. Fragment-size distributions were determined by direct visualization, using atomic force microscopy with nanometer-resolution operating in non-tapping mode, combined with an improved methodology. The fragment distributions from irradiation with gamma rays revealed discrete-like patterns at all doses, suggesting that these patterns are modulated by the base pair composition of the plasmid. Irradiation with electron beams, at very high dose rates, generated continuous distributions of highly shattered DNA fragments, similar to results at much lower dose rates found in the literature. Altogether, these results indicate that AFM could supplement traditional methods for high-resolution measurements of radiation damage to DNA, while providing new and relevant information.

In situ atomic force microscopy study of Alzheimer’s β-amyloid peptide on different substrates: New insights into mechanism of β-sheet formation

We have applied in situ atomic force microscopy to directly observe the aggregation of Alzheimer’s β-amyloid peptide (Aβ) in contact with two model solid surfaces: hydrophilic mica and hydrophobic graphite. The time course of aggregation was followed by continuous imaging of surfaces remaining in contact with 10–500 μM solutions of Aβ in PBS (pH 7.4). Visualization of fragile nanoscale aggregates of Aβ was made possible by the application of a tapping mode of imaging, which minimizes the lateral forces between the probe tip and the sample. The size and the shape of Aβ aggregates, as well as the kinetics of their formation, exhibited pronounced dependence on the physicochemical nature of the surface. On hydrophilic mica, Aβ formed particulate, pseudomicellar aggregates, which at higher Aβ concentration had the tendency to form linear assemblies, reminiscent of protofibrillar species described recently in the literature. In contrast, on hydrophobic graphite Aβ formed uniform, elongated sheets. The dimensions of those sheets were consistent with the dimensions of β-sheets with extended peptide chains perpendicular to the long axis of the aggregate. The sheets of Aβ were oriented along three directions at 120° to each other, resembling the crystallographic symmetry of a graphite surface. Such substrate-templated self-assembly may be the distinguishing feature of β-sheets in comparison with α-helices. These studies show that in situ atomic force microscopy enables direct assessment of amyloid aggregation in physiological fluids and suggest that Aβ fibril formation may be driven by interactions at the interface of aqueous solutions and hydrophobic substrates, as occurs in membranes and lipoprotein particles in vivo.

Long-term influence of chitin concentration on the resistance of cement pastes determined by atomic force microscopy

The presence of sulfates potentialize damage on cementbased materials, leading to structural failures. Therefore, structures must be designed to compensate for this effect. The mechanical properties of cement–chitin mixtures are investigated with different percentages of chitin (0.5, 1.3, and 2.1 wt.%) and aging of composite in a joint nanoscopic- and macroscopic-scale by experimental study. The objective is to increase the durability of concrete elements at coastal aquifers where concrete structures are in constant exposure to sulfate ions, chloride ions among others. Tapping mode AFM was used to characterize the surface structure and roughness of the cement pastes. To verify the chitin addition and the formation of sulfate-based aggregates Raman and IR spectra were recorded and are presented in this work. Then, force spectroscopy was used to obtain the nanomechanical properties at three different exposure times (1 day, 6 months, and 1 year) into water or a SO4 2 environment. Macroscopic parameters (e.g., compression strength of cylindrical probes) were assessed for comparison following standard guidelines. The results show a decrease of its mechanical properties as a function of the polymer concentration but more importantly, they correlate the elasticity and adhesion at the nanoscale with the behavior of the bulk material.

Caos e controle de microviga em balanço de um microscópio de força atômica, operando em modo intermitente, na ressonância

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Revealing atomic-scale details in the force fields above carbonate surfaces

Diese Arbeit stellt eine ausführliche Studie fundamentaler Eigenschaften der Kalzit CaCO3(10.4) und verwandter Mineraloberflächen dar, welche nicht nur durch die Verwendung von Nichtkontakt Rasterkraftmikroskopie, sondern hauptsächlich durch die Messung von Kraftfeldern ermöglicht wurde. Die absolute Oberflächenorientierung sowie der hierfür zugrundeliegende Prozess auf atomarer Skala konnten erfolgreich für die Kalzit (10.4) Oberfläche identifiziert werden.rnDie Adsorption chiraler Moleküle auf Kalzit ist relevant im Bereich der Biomineralisation, was ein Verständnis der Oberflächensymmetrie unumgänglich macht. Die Messung des Oberflächenkraftfeldes auf atomarer Ebene ist hierfür ein zentraler Aspekt. Eine solche Kraftkarte beleuchtet nicht nur die für die Biomineralisation wichtige Wechselwirkung der Oberfläche mit Molekülen, sondern enthält auch die Möglichkeit, Prozesse auf atomarer Skala und damit Oberflächeneigenschaften zu identifizieren.rnDie Einführung eines höchst flexiblen Messprotokolls gewährleistet die zuverlässige und kommerziell nicht erhältliche Messung des Oberflächenkraftfeldes. Die Konversion der rohen ∆f Daten in die vertikale Kraft Fz ist jedoch kein trivialer Vorgang, insbesondere wenn Glätten der Daten in Frage kommt. Diese Arbeit beschreibt detailreich, wie Fz korrekt für die experimentellen Bedingungen dieser Arbeit berechnet werden können. Weiterhin ist beschrieben, wie Lateralkräfte Fy und Dissipation Γ erhalten wurden, um das volle Potential dieser Messmethode auszureizen.rnUm Prozesse auf atomarer Skala auf Oberflächen zu verstehen sind die kurzreichweitigen, chemischen Kräfte Fz,SR von größter Wichtigkeit. Langreichweitige Beiträge müssen hierzu an Fz angefittet und davon abgezogen werden. Dies ist jedoch eine fehleranfällige Aufgabe, die in dieser Arbeit dadurch gemeistert werden konnte, dass drei unabhängige Kriterien gefunden wurden, die den Beginn zcut von Fz,SR bestimmen, was für diese Aufgabe von zentraler Bedeutung ist. Eine ausführliche Fehleranalyse zeigt, dass als Kriterium die Abweichung der lateralen Kräfte voneinander vertrauenswürdige Fz,SR liefert. Dies ist das erste Mal, dass in einer Studie ein Kriterium für die Bestimmung von zcut gegeben werden konnte, vervollständigt mit einer detailreichen Fehleranalyse.rnMit der Kenntniss von Fz,SR und Fy war es möglich, eine der fundamentalen Eigenschaften der CaCO3(10.4) Oberfläche zu identifizieren: die absolute Oberflächenorientierung. Eine starke Verkippung der abgebildeten Objekte

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.