Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: The “molecular-sandwich” technique

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of ≥0.2 nm (approximately 5% of the molecule’s height) with a time resolution of ≥1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO2) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM “molecular-sandwich” technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.

Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Note: Design and development of an integrated three-dimensional scanner for atomic force microscopy

A compact scanning head for the Atomic Force Microscope (AFM) greatly enhances the portability of AFM and facilitates easy integration with other tools. This paper reports the design and development of a three-dimensional (3D) scanner integrated into an AFM micro-probe. The scanner is realized by means of a novel design for the AFM probe along with a magnetic actuation system. The integrated scanner, the actuation system, and their associated mechanical mounts are fabricated and evaluated. The experimentally calibrated actuation ranges are shown to be over 1 mu m along all the three axes. (c) 2013 AIP Publishing LLC.

Design and Evaluation of Torsional Probes for Multifrequency Atomic Force Microscopy

Multifrequency atomic force microscopy is a powerful nanoscale imaging and characterization technique that involves excitation of the atomic force microscope (AFM) probe and measurement of its response at multiple frequencies. This paper reports the design, fabrication, and evaluation of AFM probes with a specified set of torsional eigen-frequencies that facilitate enhancement of sensitivity in multifrequency AFM. A general approach is proposed to design the probes, which includes the design of their generic geometry, adoption of a simple lumped-parameter model, guidelines for determination of the initial dimensions, and an iterative scheme to obtain a probe with the specified eigen-frequencies. The proposed approach is employed to design a harmonic probe wherein the second and the third eigen-frequencies are the corresponding harmonics of the first eigen-frequency. The probe is subsequently fabricated and evaluated. The experimentally evaluated eigen-frequencies and associated mode shapes are shown to closely match the theoretical results. Finally, a simulation study is performed to demonstrate significant improvements in sensitivity to the second-and the third-harmonic spectral components of the tip-sample interaction force with the harmonic probe compared to that of a conventional probe.

Direct Measurement of Three-Dimensional Forces in Atomic Force Microscopy

Direct measurement of three-dimensional (3-D) forces between an atomic force microscope (AFM) probe and the sample benefits diverse applications of AFM, including force spectroscopy, nanometrology, and manipulation. This paper presents the design and evaluation of a measurement system, wherein the deflection of the AFM probe is obtained at two points to enable direct measurement of all the three components of 3-D tip-sample forces in real time. The optimal locations for measurement of deflection on the probe are derived for a conventional AFM probe. Further, a new optimal geometry is proposed for the probe that enables measurement of 3-D forces with identical sensitivity and nearly identical resolution along all three axes. Subsequently, the designed measurement system and the optimized AFM probe are both fabricated and evaluated. The evaluation demonstrates accurate measurement of tip-sample forces with minimal cross-sensitivities. Finally, the real-time measurement system is employed as part of a feedback control system to regulate the normal component of the interaction force, and to perform force-controlled scribing of a groove on the surface of polymethyl methacrylate.

Recent development of tip modification techniques in chemical force microscope

A review is given on the recent development of scanning probe microscope (SPM) tip modification techniques for chemical force microscope, including the preparation and application of SPM tip modified by self-assembled monolayer, atomic force microscope (AFM) tip modified by biological molecule, scanning tunneling microscope tip modified by electrochemical method, AFM tip modified by carbon nanotube.

Effect of Eu3+ on the domain structures in phosphatidylcholine LB monolayer film observed by atomic force microscopy

Ordered domain structures were observed by atomic force microscope in dipalmitoylphosphatidycholine monolayer film, which was spread on the subphase of Eu3+ solution.

Taking nanomedicine teaching into practice with atomic force microscopy and force spectroscopy

© 2015 The American Physiological Society

Mapping of adhesion forces on soil minerals in air and water by atomic force spectroscopy (AFS)

The adhesion force between an atomic force microscope (AFM) tip and sample surfaces, mica and quartz substrates, was measured in air and water. The force curves show that the adhesion has a strong dependence on both the surface roughness and the environmental conditions surrounding the sample. The variability of the adhesion force was examined in a series of measurements taken at the same point, as well as at different places on the sample surface. The adhesion maps obtained from the distribution of the measured forces indicated regions contaminated by either organic compounds or adsorbed water. Using simple mathematical expressions we could quantitatively predict the adhesion force behavior in both air and water. The experimental results are in good agreement with theoretical calculations, where the adhesion forces in air and water were mostly associated with capillary and van der Waals forces, respectively. A small long-range repulsive force is also observed in water due to the overlapping electrical double-layers formed on both the tip and sample surfaces.

Direct observation of oxidative stress on the cell wall of Saccharomyces cerevisiae strains with atomic force microscopy

We imaged pores on the surface of the cell wall of three different industrial strains of Saccharomyces cerevisiae using atomic force microscopy. The pores could be enlarged using 10 mM diamide, an SH residue oxidant that attacks surface proteins. We found that two strains showed signs of oxidative damage via changes in density and diameter of the surface pores. We found that the German strain was resistant to diamide induced oxidative damage, even when the concentration of the oxidant was increased to 50 mM. The normal pore size found on the cell walls of American strains had diameters of about 200nm. Under conditions of oxidative stress the diameters changed to 400nm.This method may prove to be a useful rapid screening process (45-60 min) to determine which strains are oxidative resistant, as well as being able to screen for groups of yeast that are sensitive to oxidative stress. This rapid screening tool may have direct applications in molecular biology (transference of the genes to inside of living cells) and biotechnology (biotransformations reactions to produce chiral synthons in organic chemistry.

Effect of dental finishing instruments on the surface roughness of composite resins as elucidated by atomic force microscopy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Forced oscillations with continuum models of atomic force microscopy

The dynamics of the AFM-atomic force microscope follows a model based in a Timoshenko cantilever beam with a tip attached at the free end and acting with the surface of a sample. General boundary conditions arise when the tip is either in contact or non-contact with the surface. The governing equations are given in matrix conservative form subject to localized loads. The eigenanalysis is done with a fundamental matrix response of a damped second-order matrix differential equation. Forced responses are found by using a Galerkin approximation of the matrix impulse response. Simulations results with harmonic and pulse forcing show the filtering character and the effects of the tip-sample interaction at the end of the beam. © 2012 American Institute of Physics.

Atomic Force Microscopy Studies of the Amyloidogenic Processes of Intrinsically Unstructured Proteins related to Neurodegenerative Diseases

Protein aggregation and formation of insoluble aggregates in central nervous system is the main cause of neurodegenerative disease. Parkinson’s disease is associated with the appearance of spherical masses of aggregated proteins inside nerve cells called Lewy bodies. α-Synuclein is the main component of Lewy bodies. In addition to α-synuclein, there are more than a hundred of other proteins co-localized in Lewy bodies: 14-3-3η protein is one of them. In order to increase our understanding on the aggregation mechanism of α-synuclein and to study the effect of 14-3-3η on it, I addressed the following questions. (i) How α-synuclein monomers pack each other during aggregation? (ii) Which is the role of 14-3-3η on α-synuclein packing during its aggregation? (iii) Which is the role of 14-3-3η on an aggregation of α-synuclein “seeded” by fragments of its fibrils? In order to answer these questions, I used different biophysical techniques (e.g., Atomic force microscope (AFM), Nuclear magnetic resonance (NMR), Surface plasmon resonance (SPR) and Fluorescence spectroscopy (FS)).

From lipid bilayers to synaptic vesicles: atomic force microscopy on lipid-based systems

The aim of this thesis was to apply the techniques of the atomic force microscope (AFM) to biological samples, namely lipid-based systems. To this end several systems with biological relevance based on self-assembly, such as a solid-supported membrane (SSM) based sensor for transport proteins, a bilayer of the natural lipid extract from an archaebacterium, and synaptic vesicles, were investigated by the AFM. For the characterization of transport proteins with SSM-sensors proteoliposomes are adsorbed that contain the analyte (transport protein). However the forces governing bilayer-bilayer interactions in solution should be repulsive under physiological conditions. I investigated the nature of the interaction forces with AFM force spectroscopy by mimicking the adsorbing proteoliposome with a cantilever tip, which was functionalized with charged alkane thiols. The nature of the interaction is indeed repulsive, but the lipid layers assemble in stacks on the SSM, which expose their unfavourable edges to the medium. I propose a model by which the proteoliposomes interact with these edges and fuse with the bilayer stacks, so forming a uniform layer on the SSM. Furthermore I characterized freestanding bilayers from a synthetic phospholipid with a phase transition at 41°C and from a natural lipid extract of the archaebacterium Methanococcus jannaschii. The synthetic lipid is in the gel-phase at room temperature and changes to the fluid phase when heated to 50°C. The bilayer of the lipid extract shows no phase transition when heated from room temperature to the growth temperature (~ 50°C) of the archeon. Synaptic vesicles are the containers of neurotransmitter in nerve cells and the synapsins are a family of extrinsic membrane proteins, that are associated with them, and believed to control the synaptic vesicle cycle. I used AFM imaging and force spectroscopy together with dynamic light scattering to investigate the influence of synapsin I on synaptic vesicles. To this end I used native, untreated synaptic vesicles and compared them to synapsin-depleted synaptic vesicles. Synapsin-depleted vesicles were larger in size and showed a higher tendency to aggregate compared to native vesicles, although their mechanical properties were alike. I also measured the aggregation kinetics of synaptic vesicles induced by synapsin I and found that the addition of synapsin I promotes a rapid aggregation of synaptic vesicles. The data indicate that synapsin I affects the stability and the aggregation state of synaptic vesicles, and confirm the physiological role of synapsins in the assembly and regulation of synaptic vesicle pools within nerve cells.

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.