High-speed Atomic Force Microscopy Images Research Articles

Design of a Hierarchical Assembly at a Solid-Liquid Interface Using an Asymmetric Protein Needle.

Design and control of processes for a hierarchical assembly of proteins remain challenging because it requires consideration of design principles with atomic-level accuracy. Previous studies have adopted symmetry-based strategies to minimize the complexity of protein-protein interactions and this has placed constraints on the structures of the resulting protein assemblies. In the present work, we used an anisotropic-shaped protein needle, gene product 5 (gp5) from bacteriophage T4 with a C-terminal hexahistidine-tag (His-tag) (gp5_CHis), to construct a hierarchical assembly with two distinct protein-protein interaction sites. High-speed atomic force microscopy (HS-AFM) measurements reveal that it forms unique tetrameric clusters through its N-terminal head on a mica surface. The clusters further self-assemble into a monolayer through the C-terminal His-tag. The HS-AFM images and displacement analyses show that the monolayer is a network-like structure rather than a crystalline lattice. Our results expand the toolbox for constructing hierarchical protein assemblies based on structural anisotropy.

Evaluation of structural alterations in actin by computational simulation and high-speed AFM to study the mechanism of myosin force generation.

In this study, we used High-Speed Atomic Force Microscopy (HS-AFM) and computational simulation to evaluate the effect of actin oxidation on myosin force generation. For computational analysis, we implemented oxidation of certain tyrosine residues in the PDB structures of G-actin monomer and F-actin filaments. The selection of oxidized residues was based on our previous work, where oxidized residues in F-actin were identified by mass spectrometry. Computationally oxidized and non-oxidized PDB structures of the G-actin monomers were used to evaluate the electrostatic potential energy as well as for the simulation of the HS-AFM images. The comparison of the G-actin electrostatic maps revealed several spots affected by oxidation. A cross-section analysis around oxidized and non-oxidized residues in the simulated HS-AFM images of G-actin monomers revealed a shift in the height values (∼0.2 nm to 1 nm in magnitude). These differences were confirmed in the computationally oxidized F-actin structures and in experiments conducted with HS-AFM using F-actin oxidized in-vitro by an NO and O2 donor, SIN-1 (∼1.5 nm in magnitude). To better understand actin oxidation-induced impairment in contractility we determined the myosin head displacements while attached to non-oxidized or SIN-1-treated F-actin. We observed that adding myosin molecules to SIN-1-treated F-actin in the presence of ATP resulted in a change of the size of myosin head displacement. The myosin heads were able to produce short (below 1 nm), moderate (2-3 nm), and long (over 4 nm) displacements on the non-oxidized F-actin, but the frequency of long displacements was significantly decreased in the SIN-1-treated F-actin. These results suggest that oxidation decreases the pool of the weak bound myosin molecules and shortens the long displacements related to the Pi release step, thus reducing the force generation by myosin motors.

Removing the parachuting artifact using two-way scanning data in high-speed atomic force microscopy.

The high-speed atomic force microscopy (HS-AFM) is a unique and prominent method to observe structural dynamics of biomolecules at single molecule level at near-physiological condition. To achieve high temporal resolution, the probe tip scans the stage at high speed which can cause the so-called parachuting artifact in the HS-AFM images. Here, we develop a computational method to detect and remove the parachuting artifact in HS-AFM images using the two-way scanning data. To merge the two-way scanning images, we employed a method to infer the piezo hysteresis effect and to align the forward- and backward-scanning images. We then tested our method for HS-AFM videos of actin filaments, molecular chaperone, and duplex DNA. Together, our method can remove the parachuting artifact from the raw HS-AFM video containing two-way scanning data and make the processed video free from the parachuting artifact. The method is general and fast so that it can easily be applied to any HS-AFM videos with two-way scanning data.

Development of hidden Markov modeling method for molecular orientations and structure estimation from high-speed atomic force microscopy time-series images

High-speed atomic force microscopy (HS-AFM) is a powerful technique for capturing the time-resolved behavior of biomolecules. However, structural information in HS-AFM images is limited to the surface geometry of a sample molecule. Inferring latent three-dimensional structures from the surface geometry is thus important for getting more insights into conformational dynamics of a target biomolecule. Existing methods for estimating the structures are based on the rigid-body fitting of candidate structures to each frame of HS-AFM images. Here, we extend the existing frame-by-frame rigid-body fitting analysis to multiple frames to exploit orientational correlations of a sample molecule between adjacent frames in HS-AFM data due to the interaction with the stage. In the method, we treat HS-AFM data as time-series data, and they are analyzed with the hidden Markov modeling. Using simulated HS-AFM images of the taste receptor type 1 as a test case, the proposed method shows a more robust estimation of molecular orientations than the frame-by-frame analysis. The method is applicable in integrative modeling of conformational dynamics using HS-AFM data.

Functional Implications of Dynamic Structures of Intrinsically Disordered Proteins Revealed by High-Speed AFM Imaging

The unique functions of intrinsically disordered proteins (IDPs) depend on their dynamic protean structure that often eludes analysis. High-speed atomic force microscopy (HS-AFM) can conduct this difficult analysis by directly visualizing individual IDP molecules in dynamic motion at sub-molecular resolution. After brief descriptions of the microscopy technique, this review first shows that the intermittent tip-sample contact does not alter the dynamic structure of IDPs and then describes how the number of amino acids contained in a fully disordered region can be estimated from its HS-AFM images. Next, the functional relevance of a dumbbell-like structure that has often been observed on IDPs is discussed. Finally, the dynamic structural information of two measles virus IDPs acquired from their HS-AFM and NMR analyses is described together with its functional implications.

3D Generation of Multipurpose Atomic Force Microscopy Tips

In this work, 3D polymeric atomic force microscopy (AFM) tips, referred to as 3DTIPs, are manufactured with great flexibility in design and function using two‐photon polymerization. With the technology holding a great potential in developing next‐generation AFM tips, 3DTIPs prove effective in obtaining high‐resolution and high‐speed AFM images in air and liquid environments, using common AFM modes. In particular, it is shown that the 3DTIPs provide high‐resolution imaging due to their extremely low Hamaker constant, high speed scanning rates due to their low quality factor, and high durability due to their soft nature and minimal isotropic tip wear; the three important features for advancing AFM studies. It is also shown that refining the tip end of the 3DTIPs by focused ion beam etching and by carbon nanotube inclusion substantially extends their functionality in high‐resolution AFM imaging, reaching angstrom scales. Altogether, the multifunctional capabilities of 3DTIPs can bring next‐generation AFM tips to routine and advanced AFM applications, and expand the fields of high speed AFM imaging and biological force measurements.

Reconstruction of Three-Dimensional Conformations of Bacterial ClpB from High-Speed Atomic-Force-Microscopy Images

ClpB belongs to the cellular disaggretase machinery involved in rescuing misfolded or aggregated proteins during heat or other cellular shocks. The function of this protein relies on the interconversion between different conformations in its native condition. A recent high-speed-atomic-force-microscopy (HS-AFM) experiment on ClpB from Thermus thermophilus shows four predominant conformational classes, namely, open, closed, spiral, and half-spiral. Analyses of AFM images provide only partial structural information regarding the molecular surface, and thus computational modeling of three-dimensional (3D) structures of these conformations should help interpret dynamical events related to ClpB functions. In this study, we reconstruct 3D models of ClpB from HS-AFM images in different conformational classes. We have applied our recently developed computational method based on a low-resolution representation of 3D structure using a Gaussian mixture model, combined with a Monte-Carlo sampling algorithm to optimize the agreement with target AFM images. After conformational sampling, we obtained models that reflect conformational variety embedded within the AFM images. From these reconstructed 3D models, we described, in terms of relative domain arrangement, the different types of ClpB oligomeric conformations observed by HS-AFM experiments. In particular, we highlighted the slippage of the monomeric components around the seam. This study demonstrates that such details of information, necessary for annotating the different conformational states involved in the ClpB function, can be obtained by combining HS-AFM images, even with limited resolution, and computational modeling.

Adsorption of SARS-CoV-2 Spike Protein S1 at Oxide Surfaces Studied by High-Speed Atomic Force Microscopy.

The ongoing coronavirus disease 2019 (COVID‐19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) represents a serious threat to the health of millions of people. Respiratory viruses such as SARS‐CoV‐2 can be transmitted via airborne and fomite routes. The latter requires virion adsorption at abiotic surfaces and most likely involves the SARS‐CoV‐2 spike protein subunit 1 (S1), which is the outermost point of its envelope. Understanding S1 spike protein interaction with fomite surfaces thus represents an important milestone on the road to fighting the spread of COVID‐19. Herein, high‐speed atomic force microscopy (HS‐AFM) is used to monitor the adsorption of the SARS‐CoV‐2 spike protein S1 at Al2O3(0001) and TiO2(100) surfaces in situ. While the single‐crystalline oxide substrates are chosen to model the native surface oxides of Al‐ and Ti‐based fomites, adsorption is studied in electrolytes that mimic the pH and major ionic components of mucosal secretions and saliva, respectively. Quantitative analysis of the obtained HS‐AFM images indicates that S1 spike protein adsorption at these surfaces is mostly governed by electrostatic interactions with possible contributions from van der Waals interactions. It thus proceeds more rapidly at the TiO2(100) than at the Al2O3(0001) surface.

Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy.

Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

FLT3 Internal Tandem Duplication Quantitation and Length Analysis By Digital PCR Paired with High-Speed AFM

Introduction DNA length polymorphisms are difficult to diagnose due to their repetitive or variable elements. Within the FLT3 gene, internal tandem duplications (ITDs) range from <30 bp to >200 bp in length and are associated with poor prognosis in acute myeloid leukemia (AML). To address these challenges, we developed digital polymerase chain reaction (dPCR) followed by high-speed atomic force microscopy (HSAFM) as a high-throughput, single-molecule approach for quantifying and sizing length polymorphisms associated with FLT3-ITDs. Methods In the first step of our approach, dPCR (Fig. 1A), a mixed sample is diluted and partitioned into micro-reactions that are individually amplified, mitigating amplification bias and producing homogeneous solutions of either wild-type (WT) or FLT3-ITD variant amplicons. We then used HSAFM to directly image individual amplicons and determine their lengths with nanoscale resolution (Fig. 1B). Length distributions were analyzed by Bayesian inference to identify their most likely character-WT or variant-by determining whether or not the 95% most credible range of mean lengths for each micro-reaction fell within a region of practical equivalence (ROPE) to WT length (Fig. 1C). Variant allele frequency (VAF) was then calculated as the proportion of variant-identified micro-reactions (Fig. 1D). We tested (a) synthetic and cell line DNA ranging from 30 bp to 217 bp, with VAF titrated down to 5%, and (b) clinical FLT3-ITD positive and negative samples, and compared dPCR-HSAFM results to those of a widespread clinical assay (Invivoscribe Leukostrat CDx FLT3 Mutation Assay). Results dPCR-HSAFM successfully returned the VAFs of all spiked-in FLT3-ITD samples across the admixture range of 5%-100%, and WT-only controls yielded VAFs of 0% (Fig. 1C,D). The four tested clinical samples determined as FLT3-ITD positive by the standard assay returned VAFs of 49%, 16%, 26%, and 61% by dPCR-HSAFM, while the two FLT3-ITD negative clinical samples returned VAFs of 6% and <1% (Fig. 1C). Mean variant length of control and clinical samples measured by dPCR-HSAFM agreed with the known lengths as reported by the Leukostrat assay or given by cell line documentation (Fig. 1C, horizontal lines). Conclusions By demonstrating accurate quantification and sizing of FLT3-ITD variants of clinically relevant size and VAF, dPCR-HSAFM supersedes the standard clinical assay, which does not report VAF. Since FLT3-ITD length and VAF are linked to AML outcome and are important for minimal residual disease following therapy, a test that provides both metrics is desirable. Furthermore, the speed, low cost, and flexibility of dPCR-HSAFM to detect long, repetitive insertions make it an attractive alternative to sequencing in the clinical diagnosis of FLT3-ITDs and other length polymorphisms. Figure 1. dCR-HSAFM technique and results. (A) Digital PCR of a mixed sample yields a homogeneous population of WT (blue) or variant FLT3-ITD (red) amplicons in each micro-reaction. (B) HSAFM images of DNA are traced with custom software to yield lengths of individual DNA molecules. (C) Length is used to classify each dPCR reaction as WT or variant, where each vertical line gives the 95% most credible range of mean length for a dPCR reaction and italicized numbers give the number of dPCR reactions for each sample. The four leftmost clinical samples were identified as FLT3-ITD positive by a standard clinical assay, while the two rightmost clinical samples were FLT3-ITD negative. The assay does not report VAF. (D) Spiked-in VAF v. detected VAF is shown for FLT3-ITD samples with 30 bp and 126 bp insertions, with spiked-in VAF ranging from 5%-100%. Vertical bars give the VAFs for ROPEs ± 10% of the initial ROPE width, which conveys the sensitivity of each VAF to the ROPE width. Figure 1 Disclosures No relevant conflicts of interest to declare.

Structural Dynamics of a Protein Domain Relevant to the Water-Oxidizing Complex in Photosystem II as Visualized by High-Speed Atomic Force Microscopy.

Photosystem II (PSII) is a multiprotein complex that has a function of light-driven water oxidation. The catalytic site of water oxidation is the Mn4CaO5 cluster, which is bound to the lumenal side of PSII through amino acid residues from the D1 and CP43 proteins and is further surrounded by the extrinsic proteins. In this study, we have for the first time visualized the structural dynamics of the lumenal region of a PSII core complex using high-speed atomic force microscopy (HS-AFM). The HS-AFM images of a PSII membrane fragment showed stepwise dissociation of the PsbP and PsbO extrinsic proteins. Upon subsequent destruction of the Mn4CaO5 cluster, the lumenal domain of CP43 was found to undergo a conformational fluctuation. The observed structural flexibility and conformational fluctuation of the CP43 lumenal domain are suggested to play important roles in the biogenesis of PSII and the photoassembly of the Mn4CaO5 cluster.

High-Speed Atomic Force Microscopy to Study Myosin Motility.

High-speed atomic force microscopy (HS-AFM) is a unique tool that enables imaging of protein molecules during their functional activity at sub-100ms temporal and submolecular spatial resolution. HS-AFM is suited for the study of highly dynamic proteins, including myosin motors. HS-AFM images of myosin V walking on actin filaments provide irrefutable evidence for the swinging lever arm motion propelling the molecule forward. Moreover, molecular behaviors thathave not been noticed before are also displayed on the AFM movies. This chapter describes the principle, underlying techniques and performance of HS-AFM, filmed images of myosin V, and mechanistic insights into myosin motility provided from the filmed images.

In situ observation of Cu electrodeposition and dissolution behavior on Au(111) by high speed AFM

We studied the early stages of Cu electro-deposition and -dissolution on Au(111) over a wide overpotential regime by using high speed atomic force microscopy (HS-AFM) with a high temporal resolution. The HS-AFM images revealed an instantaneous nucleation mechanism where epitaxial nuclei were created at a low cathode overpotential. Nuclei with straight edges were caused by the influence of the Au(111) lattice, whereas arched and uneven edges were associated with rapid structural fluctuations of Cu adatoms. For deposits formed at a high overpotential, many fine column-like deposits covered the whole surface plane and the number of Cu nuclei increased with the cathode overpotential. These features were related to the masstransportation. Furthermore, we observed small clusters, a few nm in size, which remained on the substrate after the rapid dissolution process.

High-Speed Atomic Force Microscopy Shows Conformational Dynamics of Ca2+/Calmodulin-Dependent Protein Kinase II

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of Ca2+ signaling in eukaryotic cells. An elevation in intracellular Ca2+ leads to binding of Ca2+ to calmodulin (CaM), which binds to and activates CaMKII. Upon activation, CaMKII has the ability of autophosphorylation, then CaMKII phosphorylates numerous target proteins and is involved in many cellular functions, including synaptic plasticity. Previous EM micrographs show that CaMKII forms a 12-meric holoenzyme that could respond to the frequency of the activating signal of Ca2+. However, the relationship between structural dynamics of CaMKII oligomers and its function is still unknown. Here we used high-speed atomic force microscopy (HS-AFM) to directly visualize conformational dynamics of CaMKII oligomers during Ca2+/CaM binding and phosphorylation. HS-AFM is a powerful technique to visualize conformational changes of proteins at a single molecular level in action under near physiological conditions [1, 2]. At a basal state, HS-AFM images of CaMKII showed a highly fluctuated structure of kinase domains, indicating that a fluctuation of kinase domains generates a space to access Ca2+/CaM to the binding site of CaMKII. Upon binding of Ca2+/CaM, neighbor kinase domains contact each other, and leads to generate more space to bind additional Ca2+/CaM. These conformational dynamics probably causes a positive cooperativity of Ca2+/CaM binding. In the phosphorylated state, kinase domains in CaMKII form a disordered structure, suggesting that a disordered structure phosphorylates other subunits within a CaMKII oligomer as a non-specific manner. Thus, a sequential HS-AFM images shows importance of structural dynamics of kinase domains in CaMKII oligomers, such as fluctuation, conformational dynamics and a transition to a disordered structure. [1] M. Shibata et al. Nat. Commun. 8, 1430 (2017). M. Shibata et al. Sci. Rep. 8, 8262 (2018).

Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.

Lyapunov Estimator for High-Speed Demodulation in Dynamic Mode Atomic Force Microscopy

In dynamic mode atomic force microscopy (AFM), the imaging bandwidth is governed by the slowest component in the open-loop chain consisting of the vertical actuator, cantilever, and demodulator. While the common demodulation method is to use a lock-in amplifier (LIA), its performance is ultimately bounded by the bandwidth of the postmixing low-pass filters. This brief proposes an amplitude and phase estimation method based on a strictly positive real Lyapunov design approach. The estimator is designed to be of low complexity while allowing for high bandwidth. In addition, suitable gains for high performance are suggested such that no tuning is necessary. The Lyapunov estimator is experimentally implemented for amplitude demodulation and shown to surpass the LIA in terms of tracking bandwidth and noise performance. High-speed AFM images are presented to corroborate the results.

X-ray crystallographic and high-speed AFM studies of peroxiredoxin 1 from Chlamydomonas reinhardtii.

Peroxiredoxins (PRXs) are a group of antioxidant enzymes that are found in all organisms, including plants and green algae. The 2-Cys PRX from Chlamydomonas reinhardtii (CrPRX1) is a chloroplast-localized protein that is critical for clearing reactive oxygen species in chloroplasts. CrPRX1 is reduced by thioredoxins or calredoxin (CrCRX), a recently identified calcium-dependent redox protein. The molecular interaction between PRXs and thioredoxin/CrCRX is functionally important, but discussion has been limited owing to a lack of structural information on CrPRX1, especially regarding its oligomeric state. In this study, high-speed atomic force microscopy (HS-AFM) images of CrPRX1 and an X-ray crystallographic analysis have enabled examination of the oligomeric state of CrPRX1. Diffraction data from a crystal of the Cys174Ser mutant of CrPRX1 indicate the existence of noncrystallographic fivefold symmetry. HS-AFM images of CrPRX1 further show that CrPRX1 particles form rings with pentagonal rotational symmetry. On the basis of these findings, the oligomeric state of CrPRX1 is discussed and it is concluded that this PRX exists in a ring-shaped decameric form comprising a pentamer of dimers.

Assembling of a Pore-Forming Toxin on a Model Membrane

The assembling of the sphingomyelin (SM)-binding pore-forming toxin (PFT), lysenin, to SM/cholesterol bilayer was examined by high-speed atomic force microscopy (HS-AFM) [1]. The HS-AFM images of SM/cholesterol bilayer preincubated with lysenin exhibited the hexagonal close packed (hcp) assembly of lysenin oligomers (Fig. 1A). The in-situ AFM images revealed that the formation of the hcp structure took place quickly (Fig. 1B). Before the full coverage of the membrane surface with a stable hcp assembly of lysenin oligomers, most of the oligomers underwent reorganization either by dissociating into monomers or by rapidly diffusing along the membrane in less than a second. The assembling of lysenin oligomers was also followed on SM/DOPC/cholesterol bilayer. Oligomers firstly formed at the edges of the SM-rich domains and covered these domains similarly to the SM/cholesterol bilayer. Our results revealed the dynamic nature of the oligomers of a lipid binding toxin during its assembling on SM-containing membranes.Reference[1] N. Yilmaz, T. Yamada, P. Greimel, T. Uchihashi, T. Ando, and T. Kobayashi, Biophys. J. 105, 1397-1405 (2013).View Large Image | View Hi-Res Image | Download PowerPoint Slide

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events.

Real-Time Visualization of a Pore-Forming Toxin Assembling on a Model Membrane

The assembly of the sphingomyelin (SM)-binding pore-forming toxin (PFT), lysenin, on SM/cholesterol bilayer was examined by high-speed atomic force microscopy (HS-AFM). Previous studies suggest that lysenin oligomerizes after binding to SM and forms a honeycomb structure. The HS-AFM images of SM/ cholesterol bilayer preincubated with lysenin exhibited the honeycomb assembly of the lysenin oligomers. The time-lapse AFM images revealed that the honeycomb formation took place quickly. During honeycomb formation most of the oligomers underwent reorganization either by dissociating into monomers or by rapidly diffusing along the membrane in less than a second. In the period of reorganization, the mobile oligomers arranged into a static, well-ordered lattice. Once this static layer was formed, the lysenin molecules were firmly bound to the SM/cholesterol bilayer and the oligomers neither dissociated nor diffused. Our results revealed the dynamic nature of the oligomers of a lipid binding toxin during honeycomb formation.