Collagen has been evolutionarily selected as the preferred building block of extracellular structures, where it constitutes approximately 20% of the body’s protein mass. Despite the inherent and surprising thermodynamic instability of individual proteins at body temperature, collagen manages to assemble into higher-order structures that provide mechanical support to tissues and are widely used as scaffolds for tissue engineering. Our studies are aimed at understanding how chemical composition, environment and mechanical force influence the thermal metastability of collagen proteins. In this talk, I will describe how we are using the techniques of atomic force microscopy and centrifuge force microscopy to study collagen’s mechanics and stability, one molecule at a time. Our work is revealing clues as to how stability is encoded within collagen’s sequence, and how collagen’s triple helix balances structural stability with responsiveness to applied force and its chemical and thermal environment.

In this Comment, we direct attention to initial efforts to establish a high-quality databank of atomic force microscopy (AFM) data: bioAFM-DB. We outline the state of this endeavor, its challenges, and potential courses of action.

Nature has used proteins to evolve molecular motors that facilitate life. Although small-molecule- and DNA-based molecular motors have been synthesized, the creation of an artificial motor protein has remained a goal of synthetic biology. Here we describe a modular approach to create an artificial motor protein, the Tumbleweed (TW). TW has three legs, each with a ligand-gated DNA binding domain that controls binding to a DNA track. TW works via a Brownian ratchet mechanism where steps are effected by diffusion and then rectified by the controlling ligands. Using single-molecule fluorescence assays and a microfluidic device, we show that TW steps directionally along a DNA track when the ligand concentrations are altered in sequence. ### Competing Interest Statement The authors have declared no competing interest.

Collagen has been evolutionarily selected as the preferred building block of extracellular structures. Despite inherent thermodynamic instability of individual proteins at body temperature, collagen manages to assemble into higher-order structures that provide mechanical support to tissues. Sequence features that enhance collagen stability have been deduced primarily from studies of collagen-mimetic peptides, as collagen's large size has precluded high-resolution studies of its structure. Thus, methods are needed to analyze the structure and mechanics of full-length collagen proteins. In this study, we used AFM imaging to investigate the thermal response of collagen type IV, a key component of basement membranes. We observed a time-dependent loss of folded structures upon exposure to body temperature, with structural destabilization along the collagenous domain reflected by shorter contour lengths (seen also for collagens type I and III). We characterized the sequence-dependent bending stiffness profile of collagen IV as a function of temperature and identified a putative initiation site for thermally induced unfolding. Interchain disulfide bonds in collagen IV were shown to enhance thermal stability and serve as primary nucleation sites for in vitro refolding. In contrast to the canonical C-to-N-terminal folding direction, we found an interchain cystine knot to enable folding in the opposite direction. A multiple sequence alignment revealed that this cystine knot is evolutionarily conserved across metazoan phyla, highlighting its significance in the stabilization of early collagen IV structures. Our findings provide mechanistic insight into the unfolding and refolding pathways of collagen IV, and how its heterogeneous sequence influences stability and mechanics.

For more than 100 years, germicidal lamps emitting 254 nm ultraviolet (UV) radiation have been used for drinking-water disinfection and surface sterilization. However, due to the carcinogenic nature of 254 nm UV, these lamps have been unable to be used for clinical procedures such as wound or surgical site sterilization. Recently, technical advances have facilitated a new generation of germicidal lamp whose emissions centre at 222 nm. These novel 222 nm lamps have commensurate antimicrobial properties to 254 nm lamps while producing few short- or long-term health effects in humans upon external skin exposure. However, to realize the full clinical potential of 222 nm UV, its safety upon internal tissue exposure must also be considered. Type I collagen is the most abundant structural protein in the body, where it self-assembles into fibrils which play a crucial role in connective tissue structure and function. In this work, we investigate the effect of 222 nm UV radiation on type I collagen fibrils in vitro . We show that collagen’s response to irradiation with 222 nm UV is fluence-dependent, ranging from no detectable fibril damage at low fluences to complete fibril degradation and polypeptide chain scission at high fluences. However, we also show that fibril degradation is significantly attenuated by increasing collagen sample thickness. Given the low fluence threshold for bacterial inactivation and the macroscopic thickness of collagenous tissues in vivo , our results suggest a range of 222 nm UV fluences which may inactivate pathogenic bacteria without causing significant damage to fibrillar collagen. This presents an initial step toward the validation of 222 nm UV radiation for internal tissue disinfection.

Inspired by biology, great progress has been made in creating artificial molecular motors. However, the dream of harnessing proteins – the building blocks selected by nature – to design autonomous motors has so far remained elusive. Here we report the synthesis and characterization of the Lawnmower, an autonomous, protein-based artificial molecular motor comprised of a spherical hub decorated with proteases. Its “burnt-bridge” motion is directed by cleavage of a peptide lawn, promoting motion towards unvisited substrate. We find that Lawnmowers exhibit directional motion with average speeds of up to 80 nm/s, comparable to biological motors. By selectively patterning the peptide lawn on microfabricated tracks, we furthermore show that the Lawnmower is capable of track-guided motion. Our work opens an avenue towards nanotechnology applications of artificial protein motors.

Abstract To design an artificial protein-based molecular motor that can autonomously step along a track is a key challenge of protein design and synthetic biology. We lay out a roadmap for how to achieve this aim, based on a modular approach that combines the use of natural, non-motor proteins with de novo design. We define what can be considered to constitute a successful artificial protein motor, identify key steps along the path to achieve these designs, and provide a vision for the future beyond this aim.

Advanced Glycation End Products (AGEs) are the end result of the irreversible, non-enzymatic glycation of proteins by reducing sugars. These chemical modifications accumulate with age and have been associated with various age-related and diabetic complications. AGEs predominantly accumulate on proteins with slow turnover rates, of which collagen is a prime example. Glycation has been associated with tissue stiffening and reduced collagen fibril remodelling. In this study, we investigate the effects of glycation on the stability of type I collagen, its molecular-level mechanics and its ability to perform its physiological role of self-assembly. Collagen AGEing is induced in vitro by incubation with ribose. We confirm and assess glycation using fluorescence measurements and changes in collagen's electrophoretic mobility. Susceptibility to trypsin digestion and circular dichroism (CD) spectroscopy are used to probe changes in collagen's triple helical stability, revealing decreased stability due to glycation. Atomic Force Microscopy (AFM) imaging is used to quantify how AGEing affects collagen flexibility, where we find molecular-scale stiffening. Finally we use microscopy to show that glycated collagen molecules are unable to self-assemble into fibrils. These findings shed light on the molecular mechanisms underlying AGE-induced tissue changes, offering insight into how glycation modifies protein structure and stability.

In the past three decades, the technology of optical tweezers has made significant contributions in various scientific areas, including optics, photonics, and nanosciences. Breakthroughs include manipulating particles in both static and dynamic ways, particle sorting, and constructing controllable micromachines. Advances in shaping and controlling the laser beam profile enable control over the position and location of the trap, which has many possible applications. A line optical tweezer (LOT) can be created by rapidly moving a spot optical tweezer using a tool such as a galvanometer mirror or an acousto-optic modulator. By manipulating the intensity profile along the beam line to be asymmetric or non-uniform, the technique can be adapted to various specific applications. Among the many exciting applications of line optical tweezers, in this work, we discuss in detail applications of LOT, including probing colloidal interactions, transporting and sorting of colloidal microspheres, self-propelled motions, trapping anisotropic particles, exploring colloidal interactions at fluid-fluid interfaces, and building optical thermal ratchets. We further discuss prospective applications in each of these areas of soft matter, including polymeric and biological soft materials.

Inspired by molecular motors in biology, there has been significant progress in building artificial molecular motors, using a number of quite distinct approaches. As the constructs become more sophisticated, there is also an increasing need to directly observe the motion of artificial motors at the nanoscale and to characterize their performance. Here, we review the most used methods that tackle those tasks. We aim to help experimentalists with an overview of the available tools used for different types of synthetic motors and to choose the method most suited for the size of a motor and the desired measurements, such as the generated force or distances in the moving system. Furthermore, for many envisioned applications of synthetic motors, it will be a requirement to guide and control directed motions. We therefore also provide a perspective on how motors can be observed on structures that allow for directional guidance, such as nanowires and microchannels. Thus, this Review facilitates the future research on synthetic molecular motors, where observations at a single-motor level and a detailed characterization of motion will promote applications.

Single-molecule imaging is widely used to determine statistical distributions of molecular properties. One such characteristic is the bending flexibility of biological filaments, which can be parameterized via the persistence length. Quantitative extraction of persistence length from images of individual filaments requires both the ability to trace the backbone of the chains in the images and sufficient chain statistics to accurately assess the persistence length. Chain tracing can be a tedious task, performed manually or using algorithms that require user input and/or supervision. Such interventions have the potential to introduce user-dependent bias into the chain selection and tracing. Here, we introduce a fully automated algorithm for chain tracing and determination of persistence lengths. Dubbed "AutoSmarTrace," the algorithm is built off a neural network, trained via machine learning to identify filaments within images recorded using atomic force microscopy. We validate the performance of AutoSmarTrace on simulated images with widely varying levels of noise, demonstrating its ability to return persistence lengths in agreement with input simulation parameters. Persistence lengths returned from analysis of experimental images of collagen and DNA agree with previous values obtained from these images with different chain-tracing approaches. Although trained on atomic-force-microscopy-like images, the algorithm also shows promise to identify chains in other single-molecule imaging approaches, such as rotary-shadowing electron microscopy and fluorescence imaging.

The object of our research is to design and measure the performance of synthetic protein constructs that mimic biological nano-motors. In a previous article we presented a persistent symmetric nano-walker design named Synthetic Kinesin Inspired Protein (SKIP). Here, we extend this design to an asymmetric, directional and processive nano-motor construct called SKIP-4R and describe and contrast the effect of a rearward conservative force and a dragged load on the motor properties of the system. Unlike kinesin, SKIP-4R's motor movement is controlled by clocked ligand pulses that activate the binding and unbinding of four different repressor proteins (RA, RB, RC, RD) to an asymmetric DNA track. To simplify future synthesis and assembly, we use a redesigned structure of SKIP (Roberta Davies, private communication) in terms of a composite of two short and two longer coiled coils (rods), each linking a different repressor RA, RB, RC or RD flexibly to a central hub. The motion of SKIP-4R was simulated using Langevin dynamics in the overdamped limit both for a rearward conservative force and a dragged load and we found that the motor both stalled and then reversed in the former case, whereas it simply stalled for large drag coefficients in the latter case. We also show that SKIP-4R can be reduced to a simpler motor (SKIP-3R) when one short rod is removed. The effects of both a rearward conservative force and a dragged load on the motion of SKIP-3R and SKIP-4R are described in detail.

Extracellular matrix mechanics influence diverse cellular functions, yet surprisingly little is known about the mechanical properties of their constituent collagens. While collagen type IV is an integral component of basement membranes, it has received far less attention than the more abundant fibrillar collagens. In this work, we used atomic force microscopy to image different collagen types and analyze their sequence-dependent mechanics. By analyzing their flexibility in a sequence-dependent manner, we learned that discontinuities in the triple-helix-defining sequence (Gly-X-Y) in collagen IV lead to a generally more flexible polymer with notable flexible “hinges” that correlate with non-helical regions. We contrast these findings by studying collagen III - a continuously triple-helical collagen - in which we found that it also displays variable flexibility along its contour, most notably possessing a high flexibility region near the matrix metalloprotease (MMP) binding site. This result represents the first demonstration of a unique mechanical signature of the MMP site along collagen and offers the opportunity to examine the interplay between sequence, thermal stability, and mechanical properties. Surprisingly, we found that proline content did not correlate with local flexibility in either collagen type. We also found that physiologically relevant changes in pH and chloride concentration did not alter the flexibility of collagen IV, indicating such environmental changes are not used to control its compaction during secretion. Furthermore, we found that collagen IV exhibits oscillatory local curvature, while collagen III displays no strong preferred curvature along its contour. Our sequence-dependent curvature results show that the collagenous domain of collagen IV is structurally different than collagen III, beyond just enhanced flexibility, and we discuss possibilities for this structural profile - it could be conveying information related to collagen IV's mechanism of assembly.

Inspired by biology, great progress has been made in creating artificial molecular motors. However, the dream of harnessing proteins - the building blocks selected by Nature - to design autonomous motors has so far remained elusive. Here we report the synthesis and characterization of the Lawnmower, an autonomous, protein-based artificial molecular motor comprised of a spherical hub decorated with proteases. Its "burnt-bridge" motion is directed by cleavage of a peptide lawn, promoting motion towards unvisited substrate. We find that Lawnmowers exhibit directional motion with average speeds of up to 80 nm/s, comparable to biological motors. By selectively patterning the peptide lawn on microfabricated tracks, we furthermore show that the Lawnmower is capable of track-guided motion. Our work opens an avenue towards nanotechnology applications of artificial protein motors.

Extracellular matrix mechanics influence diverse cellular functions, yet surprisingly little is known about the mechanical properties of their constituent collagen proteins. In particular, network-forming collagen IV, an integral component of basement membranes, has been far less studied than fibril-forming collagens. A key feature of collagen IV is the presence of interruptions in the triple-helix-defining (Gly-X-Y) sequence along its collagenous domain. Here, we used atomic force microscopy to determine the impact of sequence heterogeneity on the local flexibility of collagen IV and of the fibril-forming collagen III. Our extracted flexibility profile of collagen IV reveals that it possesses highly heterogeneous mechanics, ranging from semiflexible regions as found for fibril-forming collagens to a lengthy region of high flexibility toward its N-terminus. A simple model in which flexibility is dictated only by the presence of interruptions fit the extracted profile reasonably well, providing insight into the alignment of chains and demonstrating that interruptions, particularly when coinciding in multiple chains, significantly enhance local flexibility. To a lesser extent, sequence variations within the triple helix lead to variable flexibility, as seen along the continuously triple-helical collagen III. We found this fibril-forming collagen to possess a high-flexibility region around its matrix-metalloprotease binding site, suggesting a unique mechanical fingerprint of this region that is key for matrix remodeling. Surprisingly, proline content did not correlate with local flexibility in either collagen type. We also found that physiologically relevant changes in pH and chloride concentration did not alter the flexibility of collagen IV, indicating such environmental changes are unlikely to control its compaction during secretion. Although extracellular chloride ions play a role in triggering collagen IV network formation, they do not appear to modulate the structure of its collagenous domain.

We present here a model for multivalent diffusive transport whereby a central point-like hub is coupled to multiple feet, which bind to complementary sites on a two-dimensional landscape. The available number of binding interactions is dependent on the number of feet (multivalency) and on their allowed distance from the central hub (span). Using Monte Carlo simulations that implement the Gillespie algorithm, we simulate multivalent diffusive transport processes for 100 distinct walker designs. Informed by our simulation results, we derive an analytical expression for the diffusion coefficient of a general multivalent diffusive process as a function of multivalency, span, and dissociation constant Kd. Our findings can be used to guide the experimental design of multivalent transporters, in particular, providing insight into how to overcome trade-offs between diffusivity and processivity.

The burnt-bridges ratchet (BBR) mechanism is a model for biased molecular motion whereby a random walker destroys substrate sites as it moves, thereby inhibiting backwards stepping. Nature has been shown to employ the BBR mechanism in a variety of processes, most notably the segregation of low-copy-number plasmids and the degradation of human collagen by matrix metalloproteinases. To better understand the BBR mechanism, we developed an artificial molecular motor that is designed to rectify directional motion through the cleavage of surface-bound substrate. The substrate sites are presented to the motor as a ‘lawn’ through the tips of a dense block-copolymer brush; we therefore call our artificial motor design ‘the Lawnmower’. We have shown that our surface chemistry used for the lawn has applications for fluorescence imaging and force spectroscopy [Langmuir (2018) 34, 13550]. I will present experimental tests of the Lawnmower moving on a two-dimensional lawn and quantify the extent of its superdiffusive motion.

Multi-step assembly of individual protein building blocks is key to the formation of essential higher-order structures inside and outside of cells. Optical tweezers is a technique well suited to investigate the mechanics and dynamics of these structures at a variety of size scales. In this mini-review, we highlight experiments that have used optical tweezers to investigate protein assembly and mechanics, with a focus on the extracellular matrix protein collagen. These examples demonstrate how optical tweezers can be used to study mechanics across length scales, ranging from the single-molecule level to fibrils to protein networks. We discuss challenges in experimental design and interpretation, opportunities for integration with other experimental modalities, and applications of optical tweezers to current questions in protein mechanics and assembly.

The mean-squared displacement (MSD) is an averaged quantity widely used to assess anomalous diffusion. In many cases, such as molecular motors with finite processivity, dynamics of the system of interest produce trajectories of varying duration. Here we explore the effects of finite processivity on different measures of the MSD. We do so by investigating a deceptively simple dynamical system: a one-dimensional random walk (with equidistant jump lengths, symmetric move probabilities, and constant step duration) with an origin-directed detachment bias. By tuning the time dependence of the detachment bias, we find through analytical calculations and trajectory simulations that the system can exhibit a broad range of anomalous diffusion, extending beyond conventional diffusion to superdiffusion and even superballistic motion. We analytically determine that protocols with a time-increasing detachment lead to an ensemble-averaged velocity increasing in time, thereby providing the effective acceleration that is required to push the system above the ballistic threshold. MSD analysis of burnt-bridges ratchets similarly reveals superballistic behavior. Because superdiffusive MSDs are often used to infer biased, motor-like dynamics, these findings provide a cautionary tale for dynamical interpretation.