A kinetic molecular model of the reversible unfolding and refolding of titin under force extension.

Molecular elasticity is a physicomechanical property that is associated with a select number of polypeptides and proteins, such as the giant muscle protein, titin, and the extracellular matrix protein, tenascin. Both proteins have been the subject of atomic force microscopy (AFM), laser tweezer, and other in vitro methods for examining the effects of force extension on the globular (FNIII/Ig-like) domains that comprise each protein. In this report we present a time-dependent method for simulating AFM force extension and its effect on FNIII/Ig domain unfolding and refolding. This method treats the unfolding and refolding process as a standard three-state protein folding model (U right arrow over left arrow T right arrow over left arrow F, where U is the unfolded state, T is the transition or intermediate state, and F is the fully folded state), and integrates this approach within the wormlike chain (WLC) concept. We simulated the effect of AFM tip extension on a hypothetical titin molecule comprised of 30 globular domains (Ig or FNIII) and 25% Pro-Glu-Val-Lys (PEVK) content, and analyzed the unfolding and refolding processes as a function of AFM tip extension, extension rate, and variation in PEVK content. In general, we find that the use of a three-state protein-folding kinetic-based model and the implicit inclusion of PEVK domains can accurately reproduce the experimental force-extension curves observed for both titin and tenascin proteins. Furthermore, our simulation data indicate that PEVK domains exhibit extensibility behavior, assist in the unfolding and refolding of FNIII/Ig domains in the titin molecule, and act as a force "buffer" for the FNIII/Ig domains, particularly at low and moderate extension forces.     

Correlation between surface morphology and surface forces of protein A adsorbed on mica.

We have investigated the morphology and surface forces of protein A adsorbed on mica surface in the protein solutions of various concentrations. The force-distance curves, measured with a surface force apparatus (SFA), were interpreted in terms of two different regimens: a "large-distance" regimen in which an electrostatic double-layer force dominates, and an "adsorbed layer" regimen in which a force of steric origin dominates. To further clarify the forces of steric origin, the surface morphology of the adsorbed protein layer was investigated with an atomic force microscope (AFM) because the steric repulsive forces are strongly affected by the adsorption mode of protein A molecules on mica. At lower protein concentrations (2 ppm, 10 ppm), protein A molecules were adsorbed "side-on" parallel to the mica surfaces, forming a monolayer of approximately 2.5 nm. AFM images at higher concentrations (30 ppm, 100 ppm) showed protruding structures over the monolayer, which revealed that the adsorbed protein A molecules had one end oriented into the solution, with the remainder of each molecule adsorbed side-on to the mica surface. These extending ends of protein A overlapped each other and formed a "quasi-double layer" over the mica surface. These AFM images proved the existence of a monolayer of protein A molecules at low concentrations and a "quasi-double layer" with occasional protrusions at high concentrations, which were consistent with the adsorption mode observed in the force-distance curves.     

原子力显微镜拉伸吸附在金膜表面的短单链DNA分子

对单根DNA分子的操纵和拉伸可以直接研究DNA的弹性等力学性质. 首先通过将金沉积到云母表面制备了表面粗糙度小于0.3 nm的金膜,然后一段硫代的单链DNA (100 bases) 吸附到金膜表面. 利用原子力显微镜观察不同浓度的DNA吸附在金膜上的表面形貌. 进一步用原子力显微镜的力曲线模式拉伸DNA分子,在50％的情况下DNA可以被针尖拉伸,观察到了由于针尖和DNA分子间作用力的不同导致的多种不同力曲线.     

Indentation and adhesive probing of a cell membrane with AFM: theoretical model and experiments

In probing adhesion and cell mechanics by atomic force microscopy (AFM), the mechanical properties of the membrane have an important if neglected role. Here we theoretically model the contact of an AFM tip with a cell membrane, where direct motivation and data are derived from a prototypical ligand-receptor adhesion experiment. An AFM tip is functionalized with a prototypical ligand, SIRPalpha, and then used to probe its native receptor on red cells, CD47. The interactions prove specific and typical in force, and also show in detachment, a sawtooth-shaped disruption process that can extend over hundreds of nm. The theoretical model here that accounts for both membrane indentation as well as membrane extension in tip retraction incorporates membrane tension and elasticity as well as AFM tip geometry and stochastic disruption. Importantly, indentation depth proves initially proportional to membrane tension and does not follow the standard Hertz model. Computations of detachment confirm nonperiodic disruption with membrane extensions of hundreds of nm set by membrane tension. Membrane mechanical properties thus clearly influence AFM probing of cells, including single molecule adhesion experiments.     

"Buttonin," a unique button-shaped microtubule-associated protein (75 kD) that decorates spindle microtubule surface hexagonally

A 75-kD protein was purified from sea urchin egg microtubule proteins through gel filtration. It enhanced the polymerization of porcine brain tubulin, but was not heat-stable and did not bind to calmodulin in the presence of calcium as demonstrated by calmodulin affinity column chromatography. Rotary shadowing of the freeze-etched 75-kD protein adsorbed on mica revealed the protein to be a spherical molecule (approximately 9 nm in diameter). Quick-freeze deep-etch electron microscopy revealed that the surface of microtubules polymerized with 75-kD protein was entirely covered with hexagonally packed, round, button-like structures that were quite uniform in shape and size (approximately 9 nm) and similar to the buttons observed on microtubules of mitotic spindles in vivo or microtubules isolated from mitotic spindles. Judging from calibration studies of molecular mass by gel filtration, the 75-kD protein probably exists in a dimeric form (approximately 150 kD) in its native condition. The stoichiometry of tubulin (dimer) versus 75-kD protein (dimer) in the polymerized pellet was 3-3.4:1. Hence, we concluded that the 75-kD protein was a unique microtubule-associated protein that formed the microtubule button in vivo and in vitro. We propose to name this protein "buttonin".     

Bacteriorhodopsin folds into the membrane against an external force

Despite their crucial importance for cellular function, little is known about the folding mechanisms of membrane proteins. Recently details of the folding energy landscape were elucidated by atomic force microscope (AFM)-based single molecule force spectroscopy. Upon unfolding and extraction of individual membrane proteins energy barriers in structural elements such as loops and helices were mapped and quantified with the precision of a few amino acids. Here we report on the next logical step: controlled refolding of single proteins into the membrane. First individual bacteriorhodopsin monomers were partially unfolded and extracted from the purple membrane by pulling at the C-terminal end with an AFM tip. Then by gradually lowering the tip, the protein was allowed to refold into the membrane while the folding force was recorded. We discovered that upon refolding certain helices are pulled into the membrane against a sizable external force of several tens of picoNewton. From the mechanical work, which the helix performs on the AFM cantilever, we derive an upper limit for the Gibbs free folding energy. Subsequent unfolding allowed us to analyze the pattern of unfolding barriers and corroborate that the protein had refolded into the native state.     

Atomic Force Microscopy of Biological Membranes

Atomic force microscopy (AFM) is an ideal method to study the surface topography of biological membranes. It allows membranes that are adsorbed to flat solid supports to be raster-scanned in physiological solutions with an atomically sharp tip. Therefore, AFM is capable of observing biological molecular machines at work. In addition, the tip can be tethered to the end of a single membrane protein, and forces acting on the tip upon its retraction indicate barriers that occur during the process of protein unfolding. Here we discuss the fundamental limitations of AFM determined by the properties of cantilevers, present aspects of sample preparation, and review results achieved on reconstituted and native biological membranes.     

Atomic force microscopy visualization and measurement of the activity and physicochemical properties of single monomeric and oligomeric enzymes

An approach to measure the activity of single oligomers of the heme-containing enzyme cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22°C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule per unit time was 5 ± 2 protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made it possible to determine the integral and local Young’s modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37 ± 117 and 1 ± 3 MPa, respectively.     

Exploring the mechanical behavior of single intermediate filaments

Intermediate filaments (IFs) are structural elements of eukaryotic cells with distinct mechanical properties. Tissue integrity is severely impaired, in particular in skin and muscle, when IFs are either absent or malfunctioning due to mutations. Our knowledge on the mechanical properties of IFs is mainly based on tensile testing of macroscopic fibers and on the rheology of IF networks. At the single filament level, the only piece of data available is a measure of the persistence length of vimentin IFs. Here, we have employed an atomic force microscopy (AFM) based protocol to directly probe the mechanical properties of single cytoplasmic IFs when adsorbed to a solid support in physiological buffer environment. Three IF types were studied in vitro: recombinant murine desmin, recombinant human keratin K5/K14 and neurofilaments isolated from rat brains, which are composed of the neurofilament triplet proteins NF-L, NF-M and NF-H. Depending on the experimental conditions, the AFM tip was used to laterally displace or to stretch single IFs on the support they had been adsorbed to. Upon applying force, IFs were stretched on average 2.6-fold. The maximum stretching that we encountered was 3.6-fold. A large reduction of the apparent filament diameter was observed concomitantly. The observed mechanical properties therefore suggest that IFs may indeed function as mechanical shock absorbers in vivo.     

Structural Information, Resolution, and Noise in High-Resolution Atomic Force Microscopy Topographs

AFM has developed into a powerful tool in structural biology, providing topographs of proteins under close-to-native conditions and featuring an outstanding signal/noise ratio. However, the imaging mechanism exhibits particularities: fast and slow scan axis represent two independent image acquisition axes. Additionally, unknown tip geometry and tip-sample interaction render the contrast transfer function nondefinable. Hence, the interpretation of AFM topographs remained difficult. How can noise and distortions present in AFM images be quantified? How does the number of molecule topographs merged influence the structural information provided by averages? What is the resolution of topographs? Here, we find that in high-resolution AFM topographs, many molecule images are only slightly disturbed by noise, distortions, and tip-sample interactions. To identify these high-quality particles, we propose a selection criterion based on the internal symmetry of the imaged protein. We introduce a novel feature-based resolution analysis and show that AFM topographs of different proteins contain structural information beginning at different resolution thresholds: 10 Å (AqpZ), 12 Å (AQP0), 13 Å (AQP2), and 20 Å (light-harvesting-complex-2). Importantly, we highlight that the best single-molecule images are more accurate molecular representations than ensemble averages, because averaging downsizes the z-dimension and “blurs” structural details.Abbreviations: 2D, two-dimensional; 3D, three-dimensional; ACV, auto-correlation value; AFM, atomic force microscopy; AQP0, aquaporin-0; AQP2, aquaporin-2; AqpZ, aquaporin-Z; bR, bacteriorhodopsin; CCV, cross-correlation value; CTF, contrast transfer function; DPR, differential phase residual; EM, electron microscopy; FRC, Fourier ring correlation; FSC, Fourier shell correlation; IS, internal symmetry; LH2, light-harvesting-complex 2; RMSD, root mean-square deviation; SD, standard deviation; SNR, signal/noise ratio; SSNR, spectral signal/noise ratio     

Single Protein Molecule Mapping with Magnetic Atomic Force Microscopy

Understanding the structural organization and distribution of proteins in biological cells is of fundamental importance in biomedical research. The use of conventional fluorescent microscopy for this purpose is limited due to its relatively low spatial resolution compared to the size of a single protein molecule. Atomic force microscopy (AFM), on the other hand, allows one to achieve single-protein resolution by scanning the cell surface using a specialized ligand-coated AFM tip. However, because this method relies on short-range interactions, it is limited to the detection of binding sites that are directly accessible to the AFM tip. We developed a method based on magnetic (long-range) interactions and applied it to investigate the structural organization and distribution of endothelin receptors on the surface of smooth muscle cells. Endothelin receptors were labeled with 50-nm superparamagnetic microbeads and then imaged with magnetic AFM. Considering its high spatial resolution and ability to “see” magnetically labeled proteins at a distance of up to 150 nm, this approach may become an important tool for investigating the dynamics of individual proteins both on the cell membrane and in the submembrane space.     

Pretransition and progressive softening of bovine carbonic anhydrase II as probed by single molecule atomic force microscopy

To develop a simple method for probing the physical state of surface adsorbed proteins, we adopted the force curve mode of an atomic force microscope (AFM) to extract information on the mechanical properties of surface immobilized bovine carbonic anhydrase II under native conditions and in the course of guanidinium chloride-induced denaturation. A progressive increase in the population of individually softened molecules was probed under mildly to fully denaturing conditions. The use of the approach regime of force curves gave information regarding the height and rigidity of the molecule under compressive stress, whereas use of the retracting regime of the curves gave information about the tensile characteristics of the protein. The results showed that protein molecules at the beginning of the transition region possessed slightly more flattened and significantly more softened conformations compared with that of native molecules, but were still not fully denatured, in agreement with results based on solution studies. Thus the force curve mode of an AFM was shown to be sensitive enough to provide information concerning the different physical states of single molecules of globular proteins.     

Vesikin, a vesicle associated ATPase from squid axoplasm and optic lobe, has characteristics in common with vertebrate brain MAP 1 and MAP 2

Vesikin, a protein that can associate with squid axoplasmic vesicles or optic lobe microtubules, has been implicated as a force-generating molecule involved in microtubule-dependent vesicle transport [Gilbert and Sloboda, 1986, 1988]. Because vesikin crossreacts with an antibody to porcine brain microtubule associated protein 2 (MAP 2), studies were conducted to compare squid vesikin and brain MAPs. When taxol stabilized microtubules containing vesikin as a microtubule associated protein were incubated in the presence of ATP, vesikin dissociated from the microtubule subunit lattice. This behavior would be expected for an ATP-dependent, force generating molecule that serves as a crossbridge between vesicles and microtubules. When chick brain microtubules were treated under the same conditions, MAP 2 remained bound to the microtubules while MAP 1 dissociated in a manner similar to vesikin. One dimensional peptide mapping procedures revealed that, although digestion of vesikin and MAP 2 generated several peptides common to both proteins, vesikin and MAP 2 are clearly not identical. Furthermore, the addition of vesikin or MAPS 1 and 2 to purified tubulin stimulated microtubule assembly in a manner dependent on the concentration of added protein. These findings demonstrate that brain MAPs share characteristics common to squid vesikin and support the suggestion that brain MAPs 1 and 2 might act as a force generating complex for vesicle transport in higher organisms.     

THE ATOMIC FORCE MICROSCOPE DETECTS ATP-SENSITIVE PROTEIN CLUSTERS IN THE PLASMA MEMBRANE OF TRANSFORMED MDCK CELLS

Plasma membrane proteins are supposed to form clusters that allow ‘functional cross-talk’ between individual molecules within nanometre distance. However, such hypothetical protein clusters have not yet been shown directly in native plasma membranes. Therefore, we developed a technique to get access to the inner face of the plasma membrane of cultured transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding from the cytoplasmic membrane surface. We used the K⁺channel blocker iberiotoxin (IBTX), a positively charged toxin molecule, that binds with high affinity to plasma membrane potassium channels and to atomically flat mica. Thus, apical plasma membranes could be ‘glued’ with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out membrane patches was imaged with AFM in electrolyte solution mimicking the cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9±0.02nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20nm. Large protrusions were most likely protein clusters. Addition of the proteolytic enzyme pronase to the bath solution led to the disappearance of the proteins within minutes. The metabolic substrate ATP induced a shape-change of the protein clusters and smaller subunits became visible. ADP or the non-hydrolysable ATP analogue, ATP-γ-S, could not exert similar effects. It is concluded that plasma membrane proteins (and/or membrane associated proteins) form ‘functional clusters’ in their native environment. The ‘physiological’ arrangement of the protein molecules within a cluster requires ATP.     

Examination of surface-bound Ku-DNA complexes in an aqueous environment using MAC mode atomic force microscopy

In the development of biosensors, it is essential to understand how the signal-transducing element may perturb surface-bound proteins and nucleic acids. The tip of the atomic force microscope is such an element in atomic force microscopy. In this paper, we describe the influence of tip-sample interactions on the measured height of the DNA repair protein, Ku, that has been adsorbed onto a mica surface which was submerged in aqueous solution. We find that the measured height of the Ku molecule depends critically on whether or not it is associated with DNA. Additionally, we observed that the conditions (time and concentration) under which Ku is incubated with DNA, affect the appearance (number and type) of the DNA-Ku complexes observed.     

Atomic force microscopy of RecA--DNA complexes using a carbon nanotube tip

We report high resolution images of RecA-double stranded (ds) DNA complexes obtained by atomic force microscopy (AFM). When a carbon nanotube (CNT) tip was used, AFM images visualized the 10-nm pitch of RecA-dsDNA complexes and RecA filaments as three-dimensional surface topography without reconstruction analysis. The depth of the notch between two pitches was less than 1 nm. When adsorbed on a soft surface covered with proteins, naked DNA, RecA monomers, RecA hexamers, and short RecA filaments were all clearly resolved in one image. The high resolution images with a CNT tip provided valuable information on the initiation process of RecA-dsDNA complex formation.     

肌动蛋白的原子力显微镜研究

原子力显微镜 (AFM )是一种能够在生理条件下对生物大分子、活细胞表面以及细胞膜下结构进行在体或离体研究的强有力的新型工具 ,具有原子级的成像分辨率和纳牛顿级的力测定功能。目前原子力显微镜已被广泛地应用于生物大分子、超分子体系的结构解析、动力学过程观察 ,分子力学研究及细胞功能鉴定。原子力显微镜能够通过尖锐探针扫描待测样品表面 ,收集被测样品表面地貌坐标数据从而对单分子或细胞进行成像或操作 ,并能通过移动探针、记录探针与样品之间的作用力 ,对生物大分子 (蛋白质、核酸和多糖等 )的结构力学特性进行分析以获取分子构象、功能及其相互关系的有用信息。肌动蛋白是一种细胞内普遍存在 ,具有广泛、复杂生理功能的重要蛋白质 ,原子力显微镜的各项功能已广泛地用于肌动蛋白结构、功能及动力学研究。通过综述原子力显微镜在肌动蛋白研究中的应用 ,阐明了原子力显微镜在现代生命科学研究中的重要意义及巨大应用前景。     

Synthesis and characterization of a photocleavable cross-linker and its application on tunable surface modification and protein photodelivery

A heterobifunctional photocleavable cross-linker based on an o-nitrobenzyl ester moiety was synthesized. The cross-linker has N-hydroxysuccinimidyl and disulfide groups attached at each end and thus can anchor a protein to a gold-coated substrate surface. Steady-state spectroscopic studies suggest that the cross-linker undergoes a clean C-O fragmentation upon irradiation with a quantum yield of 0.1. Consequently, immobilized proteins (such as avidin or antibodies) on a substrate surface can be released efficiently (>95%) under UV irradiation (lambda > 300 nm) without degrading the protein functionality. We also demonstrated protein delivery via bioconjugation of protein molecules to a gold-coated atomic-force microscope (AFM) tip. When the proteins are photoreleased from the AFM tip, they are delivered to the substrate surface as protein clusters of uniform size. This has been confirmed using both AFM and fluorescence microscopy. The application of bioconjugation in this study opens a new avenue for tunable surface modification and controllable protein delivery in studies of biological systems on the nanometer scale.     

Mapping HA‐tagged protein at the surface of living cells by atomic force microscopy

Single‐molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2‐adrenergic receptor (β2‐AR), a G protein‐coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2‐AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.