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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. High-speed AFM imaging enables a semiquantitative, realistic description of the dynamic structure of intrinsically disordered proteins.

Receptor–ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information on the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin–biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket, while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and ratedependent induced-fit motions for intermediates, which might be relevant for other receptor–ligand bonds.

The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na⁺ ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of ∼0.5 nm, ∼1.0 nm, and ∼1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter GltPh. Since CitS and GltPh are structurally unrelated, we conclude that the three-state elevators have evolved independently.

Assembly of biomolecules at solid–water interfaces requires molecules to traverse complex orientation-dependent energy landscapes through processes that are poorly understood, largely due to the dearth of in situ single-molecule measurements and statistical analyses of the rotational dynamics that define directional selection. Emerging capabilities in high-speed atomic force microscopy and machine learning have allowed us to directly determine the orientational energy landscape and observe and quantify the rotational dynamics for protein nanorods on the surface of muscovite mica under a variety of conditions. Comparisons with kinetic Monte Carlo simulations show that the transition rates between adjacent orientation-specific energetic minima can largely be understood through traditional models of in-plane Brownian rotation across a biased energy landscape, with resulting transition rates that are exponential in the energy barriers between states. However, transitions between more distant angular states are decoupled from barrier height, with jump-size distributions showing a power law decay that is characteristic of a nonclassical Levy-flight random walk, indicating that large jumps are enabled by alternative modes of motion via activated states. The findings provide insights into the dynamics of biomolecules at solid–liquid interfaces that lead to self-assembly, epitaxial matching, and other orientationally anisotropic outcomes and define a general procedure for exploring such dynamics with implications for hybrid biomolecular–inorganic materials design.

The exquisite organization exhibited by hybrid biomolecular–inorganic materials in nature has inspired the development of synthetic analogues for numerous applications. Nevertheless, a mechanistic picture of the energetic controls and response dynamics leading to organization is lacking. Furthermore, we pair high-speed atomic force microscopy with machine learning and Monte Carlo simulations to analyze the rotational dynamics of rod-like proteins on a crystal lattice, simultaneously quantifying the orientational energy landscape and transition probabilities between energetically favorable orientations. Although rotations largely follow Brownian diffusion, proteins often make large jumps in orientation, thus rapidly overcoming barriers that usually inhibit rotation. Moreover, the rotational dynamics can be tuned via protein size and solution chemistry, providing tools for controlling biomolecular assembly at inorganic interfaces.

P-glycoprotein (P-gp) is an ATP-driven transporter that effluxes a wide range of xenobiotics from cells, and its overexpression is a primary cause of multidrug resistance (MDR) in cancer. It is well-established that P-gp functions through conformational changes, yet its large-scale structural dynamics at work have been unexplored. Here, we directly visualized single P-gp molecules reconstituted in nanodiscs using high-speed atomic force microscopy (HS-AFM). The HS-AFM movies revealed that P-gp is intrinsically dynamic in its apo state, with its nucleotide-binding domains (NBDs) undergoing large, spontaneous opening and closing motions. However, addition of ATP stabilized a conformation characterized by NBD proximity with a strong tendency toward closure. We then leveraged this dynamic viewpoint to elucidate the relationship between Elacridar’s function and the resulting structural dynamics of P-gp. Elacridar is designed to overcome multidrug resistance (MDR) in cancer and acts as a potent dual inhibitor of both P-gp and the Breast Cancer Resistance Protein (BCRP), effectively blocking the drug efflux function of these transporters. This inhibitor has suggested concentration-dependent function: it is effluxed as a substrate at low concentrations and acts as an inhibitor at high concentrations. Our direct observations revealed that low concentrations induced active dynamics in P-gp, whereas high concentrations severely restricted its motion, leading to a rigid, non-productive state. Our study provides critical insights into how observing molecular motion itself can unravel complex biological mechanisms.

Dynamin is a dimeric GTPase that assembles into a helix around the neck of endocytic buds. Upon GTP hydrolysis, dynamin breaks these necks, a reaction called membrane fission. Fission requires dynamin to first constrict the membrane. It is unclear, however, how dynamin helix constriction works. Here we undertake a direct high-speed atomic force microscopy imaging analysis to visualize the constriction of single dynamin-coated membrane tubules. We show GTP-induced dynamic rearrangements of the dynamin helix turns: the average distances between turns reduce with GTP hydrolysis. These distances vary, however, over time because helical turns were observed to transiently pair and dissociate. At fission sites, these cycles of association and dissociation were correlated with relative lateral displacement of the turns and constriction. Our findings show relative longitudinal and lateral displacements of helical turns related to constriction. Our work highlights the potential of high-speed atomic force microscopy for the observation of mechanochemical proteins onto membranes during action at almost molecular resolution.

Individual Alzheimer’s disease (AD) patients have been shown to have structurally distinct amyloid-β (Aβ) aggregates, including fibrils, in their brain. These findings suggest the possibility of a relationship between AD progression and Aβ fibril structures. Thus, the characterization of the structural dynamics of Aβ could aid the development of novel therapeutic strategies and diagnosis. Protein structure and dynamics have typically been studied separately. Most of the commonly used biophysical approaches are limited in providing substantial details regarding the combination of both structure and dynamics. On the other hand, high-speed atomic force microscopy (HS-AFM), which simultaneously visualizes an individual protein structure and its dynamics in liquid in real time, can uniquely link the structure and the kinetic details, and it can also unveil novel insights. Although amyloidogenic proteins generate heterogeneously aggregated species, including transient unstable states during the aggregation process, HS-AFM elucidated the structural dynamics of individual aggregates in real time in liquid without purification and isolation. Here, we review and discuss the HS-AFM imaging of amyloid aggregation and strategies to optimize the experiments showing findings from Aβ and amylin, which is associated with type II diabetes, shares some common biological features with Aβ, and is reported to be involved in AD.

Significance Preexisting secretory IgA (S-IgA) antibodies can provide immediate immunity via their unique capability to eliminate a pathogen before it passes the mucosal barrier. Several clinical trials have reported that S-IgA against influenza virus was induced by intranasal administration of an inactivated influenza vaccine. S-IgA in mucosa consists predominantly of dimers rather than tetramers, as revealed by ultracentrifugation almost 50 years ago. However, direct evidence concerning the quaternary architectures and the physiological function of polymers larger than dimers has not been reported in healthy humans. The present study revealed, for the first time to our knowledge, the existence of large polymeric IgA in the healthy human upper respiratory mucosa, as well as the physiological functions of these molecules in protecting against viral infection in humans. Secretory IgA (S-IgA) antibodies, the major contributors to humoral mucosal immunity to influenza virus infection, are polymeric Igs present in many external secretions. In the present study, the quaternary structures of human S-IgA induced in nasal mucosa after administration of intranasal inactivated influenza vaccines were characterized in relation to neutralization potency against influenza A viruses. Human nasal IgA antibodies have been shown to contain at least five quaternary structures. Direct and real-time visualization of S-IgA using high-speed atomic force microscopy (AFM) demonstrated that trimeric and tetrameric S-IgA had six and eight antigen-binding sites, respectively, and that these structures exhibited large-scale asynchronous conformational changes while capturing influenza HA antigens in solution. Furthermore, trimeric, tetrameric, and larger polymeric structures, which are minor fractions in human nasal IgA, displayed increased neutralizing potency against influenza A viruses compared with dimeric S-IgA, suggesting that the larger polymeric than dimeric forms of S-IgA play some important roles in protection against influenza A virus infection in the human upper respiratory tract.

A double-ring-shaped tetradecameric GroEL complex assists proper protein folding in cooperation with the cochaperonin GroES. The dynamic GroEL–GroES interaction reflects the allosteric intra- and inter-ring communications and the chaperonin reaction. Therefore, revealing this dynamic interaction is essential to understanding the allosteric communications and the operation mechanism of GroEL. Nevertheless, how this interaction proceeds in the chaperonin cycle has long been controversial. Here, we directly image the dynamic GroEL–GroES interaction under conditions with and without foldable substrate protein using high-speed atomic force microscopy. Then, the imaging results obtained under these conditions and our previous results in the presence of unfoldable substrate are compared. The molecular movies reveal that the entire reaction pathway is highly complicated but basically identical irrespective of the substrate condition. A prominent (but moderate) difference is in the population distribution of intermediate species: symmetric GroEL : GroES2 and asymmetric GroEL : GroES1 complexes, and GroES–unbound GroEL. This difference is mainly attributed to the longer lifetime of GroEL : GroES1 complexes in the presence of foldable substrate. Moreover, the inter-ring communication, which is the basis for the alternating action of the two rings, occurs at two distinct (GroES association and dissociation) steps in the main reaction pathway, irrespective of the substrate condition. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.