HS-AFM single-molecule structural biology uncovers basis of transporter wanderlust kinetics.

The Pyrococcus horikoshii amino acid transporter GltPh revealed, like other channels and transporters, activity mode switching, previously termed wanderlust kinetics. Unfortunately, to date, the basis of these activity fluctuations is not understood, probably due to a lack of experimental tools that directly access the structural features of transporters related to their instantaneous activity. Here, we take advantage of high-speed atomic force microscopy, unique in providing simultaneous structural and temporal resolution, to uncover the basis of kinetic mode switching in proteins. We developed membrane extension membrane protein reconstitution that allows the analysis of isolated molecules. Together with localization atomic force microscopy, principal component analysis and hidden Markov modeling, we could associate structural states to a functional timeline, allowing six structures to be solved from a single molecule, and an inward-facing state, IFSopen-1, to be determined as a kinetic dead-end in the conformational landscape. The approaches presented on GltPh are generally applicable and open possibilities for time-resolved dynamic single-molecule structural biology.

Severe Inflammatory Idiopathic Multicentric Castleman's Disease Coexisting with Advanced Renal Cancer: A Case Report.

The present case study was conducted on a 74-year-old man who visited our department due to a left renal and retroperitoneal tumor on computed tomography (CT). The patient was diagnosed with left renal cancer lymph node metastasis and was hospitalized a few weeks prior to surgery due to fever, malaise, and severe appetite loss. Biochemical laboratory findings at admission showed markedly high levels of inflammation. The cause of high inflammatory response was paraneoplastic syndrome. Tumor resection was considered necessary, and left nephrectomy and lymphadenectomy were performed; however, it did not improve the inflammatory response. After operation, positron emission tomography-CT revealed hyperaccumulation of 18F-fluorodeoxyglucose in the bone marrow throughout the body. Pathological examination of the resected specimen and bone marrow aspiration revealed the coexistence of idiopathic multicentric Castleman disease (CD) and renal cancer. Prednisolone and tocilizumab were administered for idiopathic multicentric CD and a tyrosine kinase inhibitor for renal cancer; however, they had poor therapeutic effect, and the patient died. CD is characterized by systemic symptoms due to the overproduction of interleukin-6. Treatment for idiopathic multicentric CD involves steroid and anti-interleukin-6 therapy. The diagnostic criteria for CD require the exclusion of malignant tumors although there are some cases in which CD and malignant tumors coexist. The prognosis for CD is relatively good; however, as in this case, the prognosis of CD coexisting with uncontrollable renal cancer is insufficient due to poor improvement in the inflammatory response.

Single molecule kinetics of bacteriorhodopsin by HS-AFM.

Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s-1. The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended.

Force-induced conformational changes in PIEZO1.

PIEZO1 is a mechanosensitive channel that converts applied force into electrical signals. Partial molecular structures show that PIEZO1 is a bowl-shaped trimer with extended arms. Here we use cryo-electron microscopy to show that PIEZO1 adopts different degrees of curvature in lipid vesicles of different sizes. We also use high-speed atomic force microscopy to analyse the deformability of PIEZO1 under force in membranes on a mica surface, and show that PIEZO1 can be flattened reversibly into the membrane plane. By approximating the absolute force applied, we estimate a range of values for the mechanical spring constant of PIEZO1. Both methods of microscopy demonstrate that PIEZO1 can deform its shape towards a planar structure. This deformation could explain how lateral membrane tension can be converted into a conformation-dependent change in free energy to gate the PIEZO1 channel in response to mechanical perturbations.

A novel phase-shift-based amplitude detector for a high-speed atomic force microscope.

In any atomic force microscope operated in amplitude modulation mode, aka "tapping mode" or "oscillating mode," the most crucial operation is the detection of the cantilever oscillation amplitude. Indeed, it is the change in the cantilever oscillation amplitude that drives the feedback loop, and thus, the accuracy and speed of amplitude detection are of utmost importance for improved atomic force microscopy operation. This becomes even more crucial for the operation of a high-speed atomic force microscope (HS-AFM), where feedback operation on a single or a low number of cantilever oscillation cycles between 500 kHz and 1000 kHz oscillation frequency is desired. So far, the amplitude detection was performed by Fourier analysis of each oscillation, resulting in a single output amplitude value at the end of each oscillation cycle, i.e., 360° phase delay. Here, we present a novel analog amplitude detection circuit with theoretic continuous amplitude detection at 90° phase delay. In factual operation, when exposed to an abrupt amplitude change, our novel amplitude detector circuit reacted with a phase delay of ∼138° compared with the phase delay of ∼682° achieved by the Fourier analysis method. Integrated to a HS-AFM, the novel amplitude detector should allow faster image acquisition with lower invasiveness due to the faster and more accurate detection of cantilever oscillation amplitude change.

High-Speed Atomic Force Microscopy Reveals the Inner Workings of the MinDE Protein Oscillator.

The MinDE protein system from E. coli has recently been identified as a minimal biological oscillator, based on two proteins only: The ATPase MinD and the ATPase activating protein MinE. In E. coli, the system works as the molecular ruler to place the divisome at midcell for cell division. Despite its compositional simplicity, the molecular mechanism leading to protein patterns and oscillations is still insufficiently understood. Here we used high-speed atomic force microscopy to analyze the mechanism of MinDE membrane association/dissociation dynamics on isolated membrane patches, down to the level of individual point oscillators. This nanoscale analysis shows that MinD association to and dissociation from the membrane are both highly cooperative but mechanistically different processes. We propose that they represent the two directions of a single allosteric switch leading to MinD filament formation and depolymerization. Association/dissociation are separated by rather long apparently silent periods. The membrane-associated period is characterized by MinD filament multivalent binding, avidity, while the dissociated period is defined by seeding of individual MinD. Analyzing association/dissociation kinetics with varying MinD and MinE concentrations and dependent on membrane patch size allowed us to disentangle the essential dynamic variables of the MinDE oscillation cycle.

High-frequency microrheology reveals cytoskeleton dynamics in living cells.

Living cells are viscoelastic materials, with the elastic response dominating at long timescales (≳1 ms)1. At shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce2. Here, we develop high-frequency microrheology (HF-MR) to probe the viscoelastic response of living cells from 1Hz to 100 kHz. We report the viscoelasticity of different cell types and upon cytoskeletal drug treatments. At previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequency, providing a univocal mechanical fingerprint. Microrheology over a wide dynamic range up to the frequency of action of the molecular components provides a mechanistic understanding of cell mechanics.

Real-time Visualization of Phospholipid Degradation by Outer Membrane Phospholipase A using High-Speed Atomic Force Microscopy.

Phospholipases are abundant in various types of cells and compartments, where they play key roles in physiological processes as diverse as digestion, cell proliferation, and neural activation. In Gram-negative bacteria, outer membrane phospholipase A (OmpLA) is involved in outer-membrane lipid homeostasis and bacterial virulence. Although the enzymatic activity of OmpLA can be probed with an assay relying on an artificial monoacyl thioester substrate, only little is known about its activity on diacyl phospholipids. Here, we used high-speed atomic force microscopy (HS-AFM) to directly image enzymatic phospholipid degradation by OmpLA in real time. In the absence of Ca2+, reconstituted OmpLA diffused within a phospholipid bilayer without revealing any signs of phospholipase activity. Upon the addition of Ca2+, OmpLA was activated and degraded the membrane with a turnover of ~2 phospholipid molecules per second and per OmpLA dimer until most of the membrane phospholipids were hydrolyzed and the protein became tightly packed.

Influence of Japanese consumer gender and age on sensory attributes and preference (a case study on deep-fried peanuts).

BACKGROUND: Detailed exploration of sensory perception as well as preference across gender and age for a certain food is very useful for developing a vendible food commodity related to physiological and psychological motivation for food preference. Sensory tests including color, sweetness, bitterness, fried peanut aroma, textural preference and overall liking of deep-fried peanuts with varying frying time (2, 4, 6, 9, 12 and 15 min) at 150 °C were carried out using 417 healthy Japanese consumers. To determine the influence of gender and age on sensory evaluation, systematic statistical analysis including one-way analysis of variance, polynomial regression analysis and multiple regression analysis was conducted using the collected data. RESULTS: The results indicated that females were more sensitive to bitterness than males. This may affect sensory preference; female subjects favored peanuts prepared with a shorter frying time more than male subjects did. With advancing age, textural preference played a more important role in overall preference. Older subjects liked deeper-fried peanuts, which are more brittle, more than younger subjects did. CONCLUSION: In the present study, systematic statistical analysis based on collected sensory evaluation data using deep-fried peanuts was conducted and the tendency of sensory perception and preference across gender and age was clarified. These results may be useful for engineering optimal strategies to target specific segments to gain greater acceptance in the market. © 2017 Society of Chemical Industry.

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM.

Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward- and inward-facing conformations of a glutamate transporter homolog from archaebacterium Pyrococcus horikoshii, sodium/aspartate symporter GltPh, suggested the molecular basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here we present high-speed atomic force microscopy (HS-AFM) observations of membrane-reconstituted GltPh at work. HS-AFM movies provide unprecedented real-space and real-time visualization of the transport dynamics. Our results show transport mediated by large amplitude 1.85-nm "elevator" movements of the transport domains consistent with previous crystallographic and spectroscopic studies. Elevator dynamics occur in the absence and presence of sodium ions and aspartate, but stall in sodium alone, providing a direct visualization of the ion and substrate symport mechanism. We show unambiguously that individual protomers within the trimeric transporter function fully independently.

Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels.

Eukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. Despite progress in structure-function analysis, the conformational rearrangements underlying the gating of these channels are still unknown. Here, we image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. In the presence of cAMP, most channels are in a stable conformation, but a few molecules dynamically switch back and forth (blink) between at least two conformations with different heights. Upon cAMP depletion, more channels start blinking, with blinking heights increasing over time, suggestive of slow, progressive loss of ligands from the tetramer. We propose that during gating, MloK1 transitions from a set of mobile conformations in the absence to a stable conformation in the presence of ligand and that these conformations are central for gating the pore.

Temperature-Controlled High-Speed AFM: Real-Time Observation of Ripple Phase Transitions.

With nanometer lateral and Angstrom vertical resolution, atomic force microscopy (AFM) has contributed unique data improving the understanding of lipid bilayers. Lipid bilayers are found in several different temperature-dependent states, termed phases; the main phases are solid and fluid phases. The transition temperature between solid and fluid phases is lipid composition specific. Under certain conditions some lipid bilayers adopt a so-called ripple phase, a structure where solid and fluid phase domains alternate with constant periodicity. Because of its narrow regime of existence and heterogeneity ripple phase and its transition dynamics remain poorly understood. Here, a temperature control device to high-speed atomic force microscopy (HS-AFM) to observe dynamics of phase transition from ripple phase to fluid phase reversibly in real time is developed and integrated. Based on HS-AFM imaging, the phase transition processes from ripple phase to fluid phase and from ripple phase to metastable ripple phase to fluid phase could be reversibly, phenomenologically, and quantitatively studied. The results here show phase transition hysteresis in fast cooling and heating processes, while both melting and condensation occur at 24.15 °C in quasi-steady state situation. A second metastable ripple phase with larger periodicity is formed at the ripple phase to fluid phase transition when the buffer contains Ca2+ . The presented temperature-controlled HS-AFM is a new unique experimental system to observe dynamics of temperature-sensitive processes at the nanoscopic level.

High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale.

Annexins are abundant cytoplasmic proteins that can bind to negatively charged phospholipids in a Ca(2+)-dependent manner, and are known to play a role in the storage of Ca(2+) and membrane healing. Little is known, however, about the dynamic processes of protein-Ca(2+)-membrane assembly and disassembly. Here we show that high-speed atomic force microscopy (HS-AFM) can be used to repeatedly induce and disrupt annexin assemblies and study their structure, dynamics and interactions. Our HS-AFM set-up is adapted for such biological applications through the integration of a pumping system for buffer exchange and a pulsed laser system for uncaging caged compounds. We find that biochemically identical annexins (annexin V) display different effective Ca(2+) and membrane affinities depending on the assembly location, providing a wide Ca(2+) buffering regime while maintaining membrane stabilization. We also show that annexin is membrane-recruited and forms stable supramolecular assemblies within ∼5 s in conditions that are comparable to a membrane lesion in a cell. Molecular dynamics simulations provide atomic detail of the role played by Ca(2+) in the reversible binding of annexin to the membrane surface.

Automated force controller for amplitude modulation atomic force microscopy.

Atomic Force Microscopy (AFM) is widely used in physics, chemistry, and biology to analyze the topography of a sample at nanometer resolution. Controlling precisely the force applied by the AFM tip to the sample is a prerequisite for faithful and reproducible imaging. In amplitude modulation (oscillating) mode AFM, the applied force depends on the free and the setpoint amplitudes of the cantilever oscillation. Therefore, for keeping the applied force constant, not only the setpoint amplitude but also the free amplitude must be kept constant. While the AFM user defines the setpoint amplitude, the free amplitude is typically subject to uncontrollable drift, and hence, unfortunately, the real applied force is permanently drifting during an experiment. This is particularly harmful in biological sciences where increased force destroys the soft biological matter. Here, we have developed a strategy and an electronic circuit that analyzes permanently the free amplitude of oscillation and readjusts the excitation to maintain the free amplitude constant. As a consequence, the real applied force is permanently and automatically controlled with picoNewton precision. With this circuit associated to a high-speed AFM, we illustrate the power of the development through imaging over long-duration and at various forces. The development is applicable for all AFMs and will widen the applicability of AFM to a larger range of samples and to a larger range of (non-specialist) users. Furthermore, from controlled force imaging experiments, the interaction strength between biomolecules can be analyzed.

Glasslike Membrane Protein Diffusion in a Crowded Membrane.

Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion.

Active-site structure of the thermophilic Foc-subunit ring in membranes elucidated by solid-state NMR.

FoF1-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the Foc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TFoc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TFoc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TFoc oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H(+)-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D (13)C-(13)C correlation spectra of TFoc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C(α)i+1-C(α)i correlation spectrum of specifically (13)C,(15)N-labeled TFoc rings. The C(δ) chemical shift of Glu-56, which is essential for H(+) translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H(+)-locked conformation with Asn-23. The chemical shift of Asp-61 C(γ) of the E. coli c ring indicated an involvement of a water molecule in the H(+) locking, in contrast to the involvement of Asn-23 in the TFoc ring, suggesting two different means of proton storage in the c rings.

Sensory preferences among general Japanese consumers and physicochemical evaluation of deep-fried peanuts.

BACKGROUND: The development of food that satisfies consumer preferences is very important for producing commodities. In the present study, 132 Japanese consumers carried out sensory evaluation of deep-fried peanuts with varying frying times (2, 4, 6, 9, 12 and 15 min) at 150 °C, and the relationships among sensory elements and physicochemical properties were investigated. RESULT: The sensory scores for colour, bitterness, and deep-fried peanut aroma increased (darker or stronger) with frying time, whereas the sweetness score was relatively high (strong) for frying times of 2, 4, 6 and 9 min, and then decreased (weaker) with increasing frying time. Frying times of 4, 6 and 9 min scored higher in overall liking than other times. Multiple-regression analysis indicated that the overall liking score was positively correlated with sweetness (standardised regression coefficient, ß = +0.51) and deep-fried peanut aroma (ß = +0.26) scores but negatively correlated with bitterness score (ß = -0.25). Multiple-regression analysis also indicated a difference in sensory preference by gender. Sensory elements were closely related to the physicochemical properties, including the colour indexes (CIELAB colour space) and the sucrose and water contents. When L(*) (CIELAB colour space, lightness index) was 53-64 and water content was 10-30 g kg(-1), the mean overall liking score was relatively high implying acceptable fried peanut quality. CONCLUSION: Relationships among individual sensory elements were confirmed. Multiple-regression analysis indicated a strong positive correlation between sweetness and overall liking and a small difference in sensory preference by gender. Sensory evaluations can thus be expressed by physicochemical properties.

Instrumental evaluation and influence of gender and/or age among consumers on textural preference of deep-fried peanuts.

The present study was conducted to evaluate instrumental properties, to perform micro-structural analysis, and to research textural preference among Japanese general consumers (n=330) using raw and deep-fried peanuts with varying frying periods (2, 4, 6, 9, 12, and 15min) at 150°C. The relationship between consumer preference and the influence of gender and/or age among consumers regarding textural preference were evaluated. With increased frying time, the force at failure (FF) decreased from 26N to 18N, and the distance at failure (DF) decreased from 2.4mm to 1.0mm indicating increasing brittleness. A cross-sectional micro-structural image indicated increasing heterogeneity during frying. The mean preference score for 4min-, 6min-, 9min-, 12min-, and 15min-fried peanuts (3.0 to 3.4) exceeded that for 2min-fried peanuts (2.5 to 2.6), indicating that consumers liked easily broken peanuts. The tendency was more remarkable for middle-aged and elderly subjects (30years old and older) than for young subjects (15 to 29years old). Thus, textural perception may play a more important role in textural preference for older individuals than for young individuals. The Mann-Whitney's U test indicated no difference in textural preference between genders.

Atomic force microscopy studies of APOBEC3G oligomerization and dynamics.

The DNA cytosine deaminase APOBEC3G (A3G) is a two-domain protein that binds single-stranded DNA (ssDNA) largely through its N-terminal domain and catalyzes deamination using its C-terminal domain. A3G is considered an innate immune effector protein, with a natural capacity to block the replication of retroviruses such as HIV and retrotransposons. However, knowledge about its biophysical properties and mechanism of interaction with DNA are still limited. Oligomerization is one of these unclear issues. What is the stoichiometry of the free protein? What are the factors defining the oligomeric state of the protein? How does the protein oligomerization change upon DNA binding? How stable are protein oligomers? We address these questions here using atomic force microscopy (AFM) to directly image A3G protein in a free-state and in complexes with DNA, and using time-lapse AFM imaging to characterize the dynamics of A3G oligomers. We found that the formation of oligomers is an inherent property of A3G and that the yield of oligomers depends on the protein concentration. Oligomerization of A3G in complexes with ssDNA follows a similar pattern: the higher the protein concentrations the larger oligomers sizes. The specificity of A3G binding to ssDNA does not depend on stoichiometry. The binding of large A3G oligomers requires a longer ssDNA substrate; therefore, much smaller oligomers form complexes with short ssDNA. A3G oligomers dissociate spontaneously into monomers and this process primarily occurs through a monomer dissociation pathway.

Nanoscale structure and dynamics of ABOBEC3G complexes with single-stranded DNA.

The DNA cytosine deaminase APOBEC3G (A3G) is capable of blocking retrovirus replication by editing viral cDNA and impairing reverse transcription. However, the biophysical details of this host-pathogen interaction are unclear. We applied atomic force microscopy (AFM) and hybrid DNA substrates to investigate properties of A3G bound to single-stranded DNA (ssDNA). Hybrid DNA substrates included ssDNA with 5' or 3' ends attached to DNA duplexes (tail-DNA) and gap-DNA substrates, in which ssDNA is flanked by two double-stranded fragments. We found that A3G binds with similar efficiency to the 5' and 3' substrates, suggesting that ssDNA polarity is not an important factor. Additionally, we observed that A3G binds the single-stranded region of the gap-DNA substrates with the same efficiency as tail-DNA. These results demonstrate that single-stranded DNA ends are not needed for A3G binding. The protein stoichiometry does not depend on the ssDNA substrate type, but the ssDNA length modulates the stoichiometry of A3G in the complex. We applied single-molecule high-speed AFM to directly visualize the dynamics of A3G in the complexes. We were able to visualize A3G sliding and protein association-dissociation events. During sliding, A3G translocated over a 69-nucleotide ssDNA segment in <1 s. Association-dissociation events were more complex, as dimeric A3G could dissociate from the template as a whole or undergo a two-step process with monomers capable of sequential dissociation. We conclude that A3G monomers, dimers, and higher-order oligomers can bind ssDNA substrates in a manner independent of strand polarity and availability of free ssDNA ends.