Photoresponsive Supramolecular Polymers Capable of Intrachain Folding and Interchain Aggregation.

The competition between polymer chain folding and aggregation is a critical structuring process that determines the physical properties of synthetic and biopolymers. However, supramolecular polymer systems that exhibit both processes have not yet been reported. We herein introduce a system in which folded supramolecular polymers spontaneously undergo interchain aggregation due to a rearrangement in internal molecular order, converting them into crystalline aggregates. These folded supramolecular polymers slowly crystallize over the course of half a day, due to their characteristic higher-order structures. However, the photoisomerization of the trans-azobenzene incorporated into the monomer to the cis isomer leads to unfolding of the polymer, accelerating the intrachain and interchain molecular ordering to a few hours. The intermediate structures visualized by AFM demonstrate that the unfolding is coupled with interchain aggregation.

How Resilient is Wood Xylan to Enzymatic Degradation in a Matrix with Kraft Lignin?

Despite the potential of lignocellulose in manufacturing value-added chemicals and biofuels, its efficient biotechnological conversion by enzymatic hydrolysis still poses major challenges. The complex interplay between xylan, cellulose, and lignin in fibrous materials makes it difficult to assess underlying physico- and biochemical mechanisms. Here, we reduce the complexity of the system by creating matrices of cellulose, xylan, and lignin, which consists of a cellulose base layer and xylan/lignin domains. We follow enzymatic degradation using an endoxylanase by high-speed atomic force microscopy and surface plasmon resonance spectroscopy to obtain morphological and kinetic data. Fastest reaction kinetics were observed at low lignin contents, which were related to the different swelling capacities of xylan. We demonstrate that the complex processes taking place at the interfaces of lignin and xylan in the presence of enzymes can be monitored in real time, providing a future platform for observing phenomena relevant to fiber-based systems.

Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status.

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b' and a' domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b' and a' domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

Single-molecule level dynamic observation of disassembly of the apo-ferritin cage in solution.

The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications.

Structural properties determining low K+ affinity of the selectivity filter in the TWIK1 K+ channel.

Canonical K+ channels are tetrameric and highly K+-selective, whereas two-pore-domain K+ (K2P) channels form dimers, but with a similar pore architecture. A two-pore-domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na+ and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange-induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K+-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K+ ions, WT and both variants exhibit an amide-I band at 1680 cm-1 This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K+, a feature very similar to that of the canonical K+ channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K+ affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm-1 band upon changes from K+ to Rb+ or Cs+ conditions. High-speed atomic force microscopy disclosed that TWIK1's surface morphology largely does not change in K+ and Na+ solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful "molecular fingerprints" for assessing the properties of other K+ channels.

Combining adhesive contact mechanics with a viscoelastic material model to probe local material properties by AFM.

Viscoelastic properties are often measured using probe based techniques such as nanoindentation (NI) and atomic force microscopy (AFM). Rarely, however, are these methods verified. In this article, we present a method that combines contact mechanics with a viscoelastic model (VEM) composed of springs and dashpots. We further show how to use this model to determine viscoelastic properties from creep curves recorded by a probe based technique. We focus on using the standard linear solid model and the generalized Maxwell model of order 2. The method operates in the range of 0.01 Hz to 1 Hz. Our approach is suitable for rough surfaces by providing a defined contact area using plastic pre-deformation of the material. The very same procedure is used to evaluate AFM based measurements as well as NI measurements performed on polymer samples made from poly(methyl methacrylate) and polycarbonate. The results of these measurements are then compared to those obtained by tensile creep tests also performed on the same samples. It is found that the tensile test results differ considerably from the results obtained by AFM and NI methods. The similarity between the AFM results and NI results suggests that the proposed method is capable of yielding results comparable to NI but with the advantage of the imaging possibilities of AFM. Furthermore, all three methods allowed a clear distinction between PC and PMMA by means of their respective viscoelastic properties.

Shape of picoliter droplets on chemically striped patterned substrates.

We studied the shape of water droplets deposited using an inkjet nozzle on a chemically striped patterned substrate consisting of alternating hydrophobic and hydrophilic stripes. The droplet dimensions are comparable to the period of the stripes, typically covering up to 13 stripes. As such, our present results bridge the gap linking two regimes previously considered: (i) droplets on single stripes and (ii) droplets covering more than 50 stripes. In line with previous work on markedly smaller water droplets, the exact deposition position is important for the final shape of the droplets. A droplet with its center deposited on a hydrophobic stripe reaches a shape that is different than when it is deposited on a hydrophilic stripe. Experimental results of different droplet configurations on the same surface are in agreement with simulations using the lattice Boltzmann model. In addition, the simulations enable a detailed analysis of droplet free energies and the volume dependence. The latter reveals scaling properties of shape parameters in terms of droplet radius scaled to the period of the stripe pattern, which have remained unexplored until now.

Measuring mechanical properties with high-speed atomic force microscopy.

High-speed atomic force microscopy (HS-AFM) is now a widely used technique to study the dynamics of single biomolecules and complex structures. In the past, it has mainly been used to capture surface topography as structural analysis, leading to important discoveries not attainable by other methods. Similar to conventional AFM, the scope of HS-AFM was recently expanded to encompass quantities beyond topography, such as the measurement of mechanical properties. This review delves into various methodologies for assessing mechanical properties, ranging from semi-quantitative approaches to precise force measurements and their corresponding sample responses. We will focus on the application to single proteins such as bridging integrator-1, ion channels such as Piezo1, complex structures such as microtubules and supramolecular fibers. In all these examples, the unique combination of quantifiable force application and high spatiotemporal resolution allows to unravel mechanisms that cannot be investigated by conventional means.

Atomic force microscopy based manipulation of graphene using dynamic plowing lithography.

Tapping mode atomic force microscopy (AFM) is employed for dynamic plowing lithography of exfoliated graphene on silicon dioxide substrates. The shape of the graphene sheet is determined by the movement of the vibrating AFM probe. There are two possibilities for lithography depending on the applied force. At moderate forces, the AFM tip only deforms the graphene and generates local strain of the order of 0.1%. For sufficiently large forces the AFM tip can hook graphene and then pull it, thus cutting the graphene along the direction of the tip motion. Electrical characterization by AFM based electric force microscopy, Kelvin probe force microscopy and conductive AFM allows us to distinguish between the truly separated islands and those still connected to the surrounding graphene.

Are quality assessments in science affected by anchoring effects? The proposal of a follow-up study.

We plan to empirically study the assessment of scientific papers within the framework of the anchoring-and-adjustment heuristic. This study is a follow-up study which is intended to answer open questions from the previous study with the same topic Bornmann (2021) and Bornmann (2023). The previous and follow-up studies address a central question in research evaluation: does bibliometrics create the social order in science it is designed to measure or does bibliometrics reflect the given social order (which is dependent on the intrinsic quality of research)? If bibliometrics creates the social order, it can be interpreted as an anchoring-and-adjustment heuristic. In the planned study, we shall undertake a survey of corresponding authors with an available email address in the Web of Science database. The authors are asked to assess the quality of articles that they cited in previous papers. The authors are randomly assigned to different experimental settings in which they receive (or not) citation information or a numerical access code to enter the survey. The control group will not receive any further numerical information. In the statistical analyses, we estimate how (strongly) the quality assessments of the cited papers are adjusted by the respondents to the anchor value (citation counts or access code). Thus, we are interested in whether possible adjustments in the assessments can not only be produced by quality-related information (citation counts), but also by numbers that are not related to quality, i.e. the access code. Strong effects of the anchors would mean that bibliometrics (or any other number) create the social order they are supposed to measure.

Anchoring effects in the assessment of papers: An empirical survey of citing authors.

In our study, we have empirically studied the assessment of cited papers within the framework of the anchoring-and-adjustment heuristic. We are interested in the question whether the assessment of a paper can be influenced by numerical information that act as an anchor (e.g. citation impact). We have undertaken a survey of corresponding authors with an available email address in the Web of Science database. The authors were asked to assess the quality of papers that they cited in previous papers. Some authors were assigned to three treatment groups that receive further information alongside the cited paper: citation impact information, information on the publishing journal (journal impact factor) or a numerical access code to enter the survey. The control group did not receive any further numerical information. We are interested in whether possible adjustments in the assessments can not only be produced by quality-related information (citation impact or journal impact), but also by numbers that are not related to quality, i.e. the access code. Our results show that the quality assessments of papers seem to depend on the citation impact information of single papers. The other information (anchors) such as an arbitrary number (an access code) and journal impact information did not play a (important) role in the assessments of papers. The results point to a possible anchoring bias caused by insufficient adjustment: it seems that the respondents assessed cited papers in another way when they observed paper impact values in the survey. We conclude that initiatives aiming at reducing the use of journal impact information in research evaluation either were already successful or overestimated the influence of this information.

Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication.

ATAD2 is a non-canonical ATP-dependent histone chaperone and a major cancer target. Despite widespread efforts to design drugs targeting the ATAD2 bromodomain, little is known about the overall structural organization and regulation of ATAD2. Here, we present the 3.1 Å cryo-EM structure of human ATAD2 in the ATP state, showing a shallow hexameric spiral that binds a peptide substrate at the central pore. The spiral conformation is locked by an N-terminal linker domain (LD) that wedges between the seam subunits, thus limiting ATP-dependent symmetry breaking of the AAA+ ring. In contrast, structures of the ATAD2-histone H3/H4 complex show the LD undocked from the seam, suggesting that H3/H4 binding unlocks the AAA+ spiral by allosterically releasing the LD. These findings, together with the discovery of an inter-subunit signaling mechanism, reveal a unique regulatory mechanism for ATAD2 and lay the foundation for developing new ATAD2 inhibitors.

Tuning kinetics to control droplet shapes on chemically striped patterned surfaces.

The typically elongated shape of droplets on chemically microstriped surfaces has been suggested to depend strongly on the kinetics during deposition. Here, we unequivocally establish the importance of impact kinetics by comparing the geometry of pico- to microliter droplets deposited from an inkjet nozzle with those obtained by conventional deposition from a syringe. For large Weber numbers, the strongly enhanced spreading during the impact in combination with direction-dependent pinning of the contact line gives rise to more spherical droplets with a low aspect ratio. The impact energy can be minimized by the prolonged firing of small picoliter droplets to form larger droplets or, as shown in the past, by using high-viscosity liquids. In the first case, the impact energy is absorbed by the liquid already present, therewith reducing the impact diameter and consequently forming markedly more elongated droplets.

Microtubule Preparation for Investigation with High-Speed Atomic Force Microscopy.

High-speed atomic force microscopy (AFM) is a versatile method that can visualize proteins and protein systems on the nanometer scale and at a temporal resolution of 100 ms. The application to microtubules can not only reveal structural information with single-tubulin resolution but can also extract mechanical information and allows to study single motor proteins walking on microtubules, among others. This chapter provides a step-by-step guide from microtubule polymerization to successful observation with high-speed AFM.

Tip-scan high-speed atomic force microscopy with a uniaxial substrate stretching device for studying dynamics of biomolecules under mechanical stress.

High-speed atomic force microscopy (HS-AFM) is a powerful tool for studying the dynamics of biomolecules in vitro because of its high temporal and spatial resolution. However, multi-functionalization, such as combination with complementary measurement methods, environment control, and large-scale mechanical manipulation of samples, is still a complex endeavor due to the inherent design and the compact sample scanning stage. Emerging tip-scan HS-AFM overcame this design hindrance and opened a door for additional functionalities. In this study, we designed a motor-driven stretching device to manipulate elastic substrates for HS-AFM imaging of biomolecules under controllable mechanical stimulation. To demonstrate the applicability of the substrate stretching device, we observed a microtubule buckling by straining the substrate and actin filaments linked by α-actinin on a curved surface. In addition, a BAR domain protein BIN1 that senses substrate curvature was observed while dynamically controlling the surface curvature. Our results clearly prove that large-scale mechanical manipulation can be coupled with nanometer-scale imaging to observe biophysical effects otherwise obscured.

Anchoring effects in the assessment of papers: The proposal for an empirical survey of citing authors.

In our planned study, we shall empirically study the assessment of cited papers within the framework of the anchoring-and-adjustment heuristic. We are interested in the question whether citation decisions are (mainly) driven by the quality of cited references. The design of our study is oriented towards the study by Teplitskiy, Duede [10]. We shall undertake a survey of corresponding authors with an available email address in the Web of Science database. The authors are asked to assess the quality of papers that they cited in previous papers. Some authors will be assigned to three treatment groups that receive further information alongside the cited paper: citation information, information on the publishing journal (journal impact factor), or a numerical access code to enter the survey. The control group will not receive any further numerical information. In the statistical analyses, we estimate how (strongly) the quality assessments of the cited papers are adjusted by the respondents to the anchor value (citation, journal, or access code). Thus, we are interested in whether possible adjustments in the assessments can not only be produced by quality-related information (citation or journal), but also by numbers that are not related to quality, i.e. the access code. The results of the study may have important implications for quality assessments of papers by researchers and the role of numbers, citations, and journal metrics in assessment processes.

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade.

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

Deformation of microtubules regulates translocation dynamics of kinesin.

Microtubules, the most rigid components of the cytoskeleton, can be key transduction elements between external forces and the cellular environment. Mechanical forces induce microtubule deformation, which is presumed to be critical for the mechanoregulation of cellular events. However, concrete evidence is lacking. In this work, with high-speed atomic force microscopy, we unravel how microtubule deformation regulates the translocation of the microtubule-associated motor protein kinesin-1, responsible for intracellular transport. Our results show that the microtubule deformation by bending impedes the translocation dynamics of kinesins along them. Molecular dynamics simulation shows that the hindered translocation of kinesins can be attributed to an enhanced affinity of kinesins to the microtubule structural units in microtubules deformed by bending. This study advances our understanding of the role of cytoskeletal components in mechanotransduction.

Recent advances in bioimaging with high-speed atomic force microscopy.

Among various microscopic techniques for characterizing protein structures and functions, high-speed atomic force microscopy (HS-AFM) is a unique technique in that it allows direct visualization of structural changes and molecular interactions of proteins without any labeling in a liquid environment. Since the development of the HS-AFM was first reported in 2001, it has been applied to analyze the dynamics of various types of proteins, including motor proteins, membrane proteins, DNA-binding proteins, amyloid proteins, and artificial proteins. This method has now become a versatile tool indispensable for biophysical research. This short review summarizes some bioimaging applications of HS-AFM reported in the last few years and novel applications of HS-AFM utilizing the unique ability of AFM to gain mechanical properties of samples in addition to structural information.

Microtubule self-healing and defect creation investigated by in-line force measurements during high-speed atomic force microscopy imaging.

Microtubules are biopolymers composed of tubulin and play diverse roles in a wide variety of biological processes such as cell division, migration and intracellular transport in eukaryotic cells. To perform their functions, microtubules are mechanically stressed and, thereby, susceptible to structural defects. Local variations in mechanical properties caused by these defects modulate their biological functions, including binding and transportation of microtubule-associated proteins. Therefore, assessing the local mechanical properties of microtubules and analyzing their dynamic response to mechanical stimuli provide insight into fundamental processes. It is, however, not trivial to control defect formation, gather mechanical information at the same time, and subsequently image the result at a high temporal resolution at the molecular level with minimal delay. In this work, we describe the so-called in-line force curve mode based on high-speed atomic force microscopy. This method is directly applied to create defects in microtubules at the level of tubulin dimers and monitor the following dynamic processes around the defects. Furthermore, force curves obtained during defect formation provide quantitative mechanical information to estimate the bonding energy between tubulin dimers.