FliL ring enhances the function of periplasmic flagella.

SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of Borrelia burgdorferi, revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.

Light-Powered Propeller-like Transporter for Boosted Transmembrane Ion Transport.

Membrane-active molecular machines represent a recently emerging, yet important line of expansion in the field of artificial transmembrane transporters. Their hitherto demonstrated limited types (molecular swing, ion fishers, shuttlers, rotors, etc.) certainly call for new inspiring developments. Here, we report a very first motorized ion-transporting carrier-type transporter, i.e., a modularly tunable, light-powered propeller-like transporter derived from Feringa's molecular motor for consistently boosting transmembrane ion transport under continuous UV light irradiation. Based on the EC50 values, the molecular propeller-mediated ion transport activities under UV light irradiation for 300 s are 2.31, 1.74, 2.29, 2.80, and 2.92 times those values obtained without irradiation for Li+, Na+, K+, Rb+, and Cs+ ions, respectively, with EC50 value as low as 0.71 mol % for K+ ion under light irradiation.

Structure and function of ClpXP, a AAA+ proteolytic machine powered by probabilistic ATP hydrolysis.

ClpXP is an archetypical AAA+ protease, consisting of ClpX and ClpP. ClpX is an ATP-dependent protein unfoldase and polypeptide translocase, whereas ClpP is a self-compartmentalized peptidase. ClpXP is currently the only AAA+ protease for which high-resolution structures exist, the molecular basis of recognition for a protein substrate is understood, extensive biochemical and genetic analysis have been performed, and single-molecule optical trapping has allowed direct visualization of the kinetics of substrate unfolding and translocation. In this review, we discuss our current understanding of ClpXP structure and function, evaluate competing sequential and probabilistic mechanisms of ATP hydrolysis, and highlight open questions for future exploration.

Structural basis of membrane machines that traffick and attach heme to cytochromes.

We evaluate cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c synthase. These integral membrane proteins, CcsBA and CcmF/H, both covalently attach heme to cyt c, but carry it out via different mechanisms. A CcsB-CcsA complex transports heme through a channel to its external active site, where it forms two thioethers between reduced (Fe+2) heme and CysXxxXxxCysHis in cyt c. The active site is formed by a periplasmic WWD sequence and two histidines (P-His1 and P-His2). We evaluate each proposed functional domain in CcsBA cryoEM densities, exploring their presence in other CcsB-CcsA proteins from a wide distribution of organisms (e.g., from Gram positive to Gram negative bacteria to chloroplasts.) Two conserved pockets, for the first and second cysteines of CXXCH, explain stereochemical heme attachment. In addition to other universal features, a conserved periplasmic beta stranded structure, called the beta cap, protects the active site when external heme is not present. Analysis of CcmF/H, here called an oxidoreductase and cyt c synthase, addresses mechanisms of heme access and attachment. We provide evidence that CcmF/H receives Fe+3 heme from holoCcmE via a periplasmic entry point in CcmF, whereby heme is inserted directly into a conserved WWD/P-His domain from above. Evidence suggests that CcmF acts as a heme reductase, reducing holoCcmE (to Fe+2) through a transmembrane electron transfer conduit, which initiates a complicated series of events at the active site.

Aggregation-Induced Emissive Feringa-Type Motor: Toward the Dual-Functional Motor in a Single Molecular Aggregation System.

Aggregation-induced emission (AIE)allows tunable photoluminescence via the simple regulation of molecular aggregation. The research spurt along this vein has also offered tremendous opportunities for light-responsive artificial molecular machines that are to be fully explored for performing versatile functions. Herein, the study reports a light-driven Feringa-type motor, when in the appropriate aggregation state, not only demonstrates the light-activated rotary motion but emits photons with good quantum yield. A semi-quantitative TD-DFT calculation is also conducted to aid the understanding of the competitive photoluminescence and photoisomerization processes of the motor. Cytotoxicity test shows this motor possesses good biocompatibility, laying a solid foundation for applying it in the bio-environment. The results demonstrated that the engagement of the aggregation-induced emission concept and light-driven Feringa-motor can lead to the discovery of the novel motorized AIEgen, which will further stimulate the rise of more advanced molecular motors capable of executing multi-functionalities.

Mechanical control of molecular machines at an air-water interface: manipulation of molecular pliers, paddles.

Many artificial molecular machines have been synthesized, and various functions have been expressed by changing their molecular conformations. However, their structures are still simple compared with those of biomolecular machines, and more energy is required to control them. To design artificial molecular machines with more complex structures and higher functionality, it is necessary to combine molecular machines with simple movements such as components. This means that the motion of individual molecular machines must be precisely controlled and observed in various environments. At the air - water interface, the molecular orientation and conformation can be controlled with little energy as thermal fluctuations. We designed various molecular machines and controlled them using mechanical stimuli at the air - water interface. We also controlled the transfer of forces to the molecular machines in various lipid matrices. In this review, we describe molecular pliers with amphiphilic binaphthyl, molecular paddles with binuclear platinum complexes, and molecular rotors with julolidine and BODIPY that exhibit twisted intramolecular charge transfer.

This review discusses the dependence of the behaviour of molecular machines around their environment through the mechanically control of simple molecular machines at the air ­ water interface.

Bidirectional Molecular Motors by Controlling Threading and Dethreading Pathways of a Linked Rotaxane.

Artificial molecular motors have been presented as models for biological molecular motors. In contrast to the conventional artificial molecular motors that rely on covalent bond rotation, molecular motors with mechanically interlocked molecules (MIM) have attracted considerable attention owing to their ability to generate significant rotational motion by dynamically shuttling macrocyclic components. The topology of MIM-type rotational molecular motors is currently limited to catenane structures, which require intricate synthetic procedures that typically produce a low synthetic yield. In this study, we develop a novel class of MIM-type molecular motors with a rotaxane-type topology. The switching of the threading/dethreading pathways of the linked rotaxane by protecting/deprotecting the bulky stopper group and changing the solvent polarity enables a net unidirectional rotation of the molecular motor. The threading/dethreading reaction rates were quantitatively evaluated through detailed spectroscopic investigations. Repeated net unidirectional rotation and switching of the direction of rotation were also achieved. Our findings demonstrate that linked rotaxanes can serve as MIM-type molecular motors with reversible rotational direction controlled by threading/dethreading reactions. These motors hold potential as components of molecular machinery.

Beyond Single-Cycle Autonomous Molecular Machines: Light-Powered Shuttling in a Multi-Cycle Reaction Network.

Biomolecular machines autonomously convert energy into functions, driving systems away from thermodynamic equilibrium. This energy conversion is achieved by leveraging complex, kinetically asymmetric chemical reaction networks that are challenging to characterize precisely. In contrast, all known synthetic molecular systems in which kinetic asymmetry has been quantified are well described by simple single-cycle networks. Here, we report on a unique light-driven [2]rotaxane that enables the autonomous operation of a synthetic molecular machine with a multi-cycle chemical reaction network. Unlike all prior systems, the present one exploits a photoactive macrocycle, which features a different photoreactivity depending on the binding sites at which it resides. Furthermore, E to Z isomerization reverses the relative affinity of the macrocycle for two binding sites on the axle, resulting in a multi-cycle network. Building on the most recent theoretical advancements, this work quantifies kinetic asymmetry in a multi-cycle network for the first time. Our findings represent the simplest rotaxane capable of autonomous shuttling developed so far and offer a general strategy to generate and quantify kinetic asymmetry beyond single-cycle systems.

Lighting Up Bispyrene-Functionalized Chiral Molecular Muscles with Switchable Circularly Polarized Excimer Emissions.

Aiming at the further extension of the application scope of traditional molecular muscles, a novel bispyrene-functionalized chiral molecular [c2]daisy chain was designed and synthesized. Taking advantage of the unique dimeric interlocked structure of molecular [c2]daisy chain, the resultant chiral molecular muscle emits strong circularly polarized luminescence (CPL) attributed to the pyrene excimer with a high dissymmetry factor (glum) value of 0.010. More importantly, along with the solvent- or anion- induced motions of the chiral molecular muscle, the precise regulation of the pyrene stacking within its skeleton results in the switching towards either "inversed" state with sign inversion and larger glum values or "down" state with maintained handedness and smaller glum values, making it a novel multistate CPL switch. As the first example of chiral molecular muscle-based CPL switch, this proof-of-concept study not only successfully widens the application scopes of molecular muscles, but also provides a promising platform for the construction of novel smart chiral luminescent materials for practical applications.

Kinetic Asymmetry and Directionality of Nonequilibrium Molecular Systems.

Scientists have long been fascinated by the biomolecular machines in living systems that process energy and information to sustain life. The first synthetic molecular rotor capable of performing repeated 360° rotations due to a combination of photo- and thermally activated processes was reported in 1999. The progress in designing different molecular machines in the intervening years has been remarkable, with several outstanding examples appearing in the last few years. Despite the synthetic accomplishments, there remains confusion regarding the fundamental design principles by which the motions of molecules can be controlled, with significant intellectual tension between mechanical and chemical ways of thinking about and describing molecular machines. A thermodynamically consistent analysis of the kinetics of several molecular rotors and pumps shows that while light driven rotors operate by a power-stroke mechanism, kinetic asymmetry-the relative heights of energy barriers-is the sole determinant of the directionality of catalysis driven machines. Power-strokes-the relative depths of energy wells-play no role whatsoever in determining the sign of the directionality. These results, elaborated using trajectory thermodynamics and the nonequilibrium pump equality, show that kinetic asymmetry governs the response of many non-equilibrium chemical phenomena.

Structural and kinetic basis for the regulation and potentiation of Hsp104 function.

Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.

Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease.

ClpA is a hexameric double-ring AAA+ unfoldase/translocase that functions with the ClpP peptidase to degrade proteins that are damaged or unneeded. How the 12 ATPase active sites of ClpA, 6 in the D1 ring and 6 in the D2 ring, work together to fuel ATP-dependent degradation is not understood. We use site-specific cross-linking to engineer ClpA hexamers with alternating ATPase-active and ATPase-inactive modules in the D1 ring, the D2 ring, or both rings to determine if these active sites function together. Our results demonstrate that D2 modules coordinate with D1 modules and ClpP during mechanical work. However, there is no requirement for adjacent modules in either ring to be active for efficient enzyme function. Notably, ClpAP variants with just three alternating active D2 modules are robust protein translocases and function with double the energetic efficiency of ClpAP variants with completely active D2 rings. Although D2 is the more powerful motor, three or six active D1 modules are important for high enzyme processivity, which depends on D1 and D2 acting coordinately. These results challenge sequential models of ATP hydrolysis and coupled mechanical work by ClpAP and provide an engineering strategy that will be useful in testing other aspects of ClpAP mechanism.

Dithienylethene-Based Single Molecular Photothermal Linear Actuator.

By employing a mechanically controllable break junction technique, we have realized an ideal single molecular linear actuator based on dithienylethene (DTE) based molecular architecture, which undergoes reversible photothermal isomerization when subjected to UV irradiation under ambient conditions. As a result, open form (compressed, UV OFF) and closed form (elongated, UV ON) of dithienylethene-based molecular junctions are achieved. Interestingly, the mechanical actuation is achieved without changing the conductance of the molecular junction around the Fermi level over several cycles, which is an essential property required for an ideal single molecular actuator. Our study demonstrates a unique example of achieving a perfect balance between tunneling width and barrier height change upon photothermal isomerization, resulting in no change in conductance but a change in the molecular length, which results in mechanical actuation at the single molecular level.

SARS-CoV-2 monitoring by automated target-driven molecular machine-based engineering.

Biosensors based on nucleic acid-structured electrochemiluminescence are rapidly developing for medical diagnostics. Here, we build an automated DNA molecular machine on Ti3C2/polyethyleneimine-Ru(dcbpy)3 2+@Au composite, which alters the situation that a DNA molecular machine requires laying down motion tracks. We use this DNA molecular machine to transduce the target concentration information to enhance the electrochemiluminescence signal based on DNA hybridization calculations. Complex bioanalytical processes are centralized in a single nucleic acid probe unit, thus eliminating the tedious steps of laying down motion tracks required by the traditional molecular machine. We found a detection limit of 0.68 pM and a range of 1 pM to 1 nM for the analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific DNA target. Recoveries range between 96.4 and 104.8% for the analysis of SARS-CoV-2 in human saliva. Supplementary Information: The online version contains supplementary material available at 10.1007/s10311-022-01434-9.

DNA Origami-Based Single-Molecule CRISPR Machines for Spatially Resolved Searching.

Repurposing the RNA-guided endonuclease Cas9 to develop artificial CRISPR molecular machines represents a new direction toward synthetic molecular information processing. The operation of CRISPR-Cas9-based machines, nevertheless, relies on the molecular recognition of freely diffused sgRNA/Cas9, making it practically challenging to perform spatially regulated localized searching or navigation. Here, we develop a DNA origami-based single-molecule CRISPR machine that can perform spatially resolved DNA cleavage via either free or localized searching modes. When triggered at a specific site on the DNA origami with nanoscale accuracy, the free searching mode leads to searching activity that gradually decays with the distance, whereas the localized mode generates spatially-confined searching activity. Our work expands the function of CRISPR molecular machines and lays foundations to develop integrated molecular circuits and high-throughput nucleic acid detection.

Combination Cancer Treatment: Using Engineered DNAzyme Molecular Machines for Dynamic Inter- and Intracellular Regulation.

Despite the promise of combination cancer therapy, it remains challenging to develop targeted strategies that are nontoxic to normal cells. Here we report a combination therapeutic strategy based on engineered DNAzyme molecular machines that can promote cancer apoptosis via dynamic inter- and intracellular regulation. To achieve external regulation of T-cell/cancer cell interactions, we designed a DNAzyme-based molecular machine with an aptamer and an i-motif, as the MUC-1-selective aptamer allows the specific recognition of cancer cells. The i-motif is folded under the tumor acidic microenvironment, shortening the intercellular distance. As a result, T-cells are released by metal ion activated DNAzyme cleavage. To achieve internal regulation of mitochondria, we delivered another DNAzyme-based molecular machine with mitochondria-targeted peptides into cancer cells to induce mitochondria aggregation. Our strategy achieved an enhanced killing effect in zinc deficient cancer cells.

Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation.

Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, whereas the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from Drosophila melanogaster, which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.

Artificial Supramolecular Pumps Powered by Light.

The development of artificial nanoscale motors that can use energy from a source to perform tasks requires systems capable of performing directionally controlled molecular movements and operating away from chemical equilibrium. Here, the design, synthesis and properties of pseudorotaxanes are described, in which a photon input triggers the unidirectional motion of a macrocyclic ring with respect to a non-symmetric molecular axle. The photoinduced energy ratcheting at the basis of the pumping mechanism is validated by measuring the relevant thermodynamic and kinetic parameters. Owing to the photochemical behavior of the azobenzene moiety embedded in the axle, the pump can repeat its operation cycle autonomously under continuous illumination. NMR spectroscopy was used to observe the dissipative non-equilibrium state generated in situ by light irradiation. We also show that fine changes in the axle structure lead to an improvement in the performance of the motor. Such results highlight the modularity and versatility of this minimalist pump design, which provides facile access to dynamic systems that operate under photoinduced non-equilibrium regimes.

Divergent Synthesis of Molecular Winch Prototypes.

We report the synthesis of conceptually new prototypes of molecular winches with the ultimate aim to investigate the work performed by a single ruthenium-based molecular motor anchored on a surface by probing its ability to pull a load upon electrically-driven directional rotation. According to a technomimetic design, the motor was embedded in a winch structure, with a long flexible polyethylene glycol chain terminated by an azide hook to connect a variety of molecular loads. The structure of the motor was first derivatized by means of two sequential cross-coupling reactions involving a penta(4-halogenophenyl)cyclopentadienyl hydrotris(indazolyl)borate ruthenium(II) precursor and the resulting benzylamine derivative was next exploited as key intermediate in the divergent synthesis of a family of nanowinch prototypes. A one-pot method involving sequential peptide coupling and Cu-catalyzed azide-alkyne cycloaddition was developed to yield four loaded nanowinches, with load fragments encompassing triptycene, fullerene and porphyrin moieties.

Molecular rotary motors: Unidirectional motion around double bonds.

The field of synthetic molecular machines has quickly evolved in recent years, growing from a fundamental curiosity to a highly active field of chemistry. Many different applications are being explored in areas such as catalysis, self-assembled and nanostructured materials, and molecular electronics. Rotary molecular motors hold great promise for achieving dynamic control of molecular functions as well as for powering nanoscale devices. However, for these motors to reach their full potential, many challenges still need to be addressed. In this paper we focus on the design principles of rotary motors featuring a double-bond axle and discuss the major challenges that are ahead of us. Although great progress has been made, further design improvements, for example in terms of efficiency, energy input, and environmental adaptability, will be crucial to fully exploit the opportunities that these rotary motors offer.