Artem K. Efremov

Dynamics and stability of polymorphic human telomeric G-quadruplex under tension

As critical DNA structures capping the human chromosome ends, the stability and structural polymorphism of human telomeric G-quadruplex (G4) have drawn increasing attention in recent years. This work characterizes the equilibrium transitions of single-molecule telomeric G4 at physiological K+ concentration. We report three folded states of telomeric G4 with markedly different lifetime and mechanical stability. Our results show that the kinetically favored folding pathway is through a short-lived intermediate state to a longer-lived state. By examining the force dependence of transition rates, the force-dependent transition free energy landscape for this pathway is determined. In addition, an ultra-long-lived form of telomeric G4 structure with a much stronger mechanical stability is identified.

Universal optimal working cycles of molecular motors

Molecular motors capable of directional track-walking or rotation are abundant in living cells, and inspire the emerging field of artificial nanomotors. Some biomotors can convert 90% of free energy from chemical fuels into usable mechanical work, and the same motors still maintain a speed sufficient for cellular functions. This study exposed a new regime of universal optimization that amounts to a thermodynamically best working regime for molecular motors but is unfamiliar in macroscopic engines. For the ideal case of zero energy dissipation, the universally optimized working cycle for molecular motors is infinitely slow like Carnot cycle for heat engines. But when a small amount of energy dissipation reduces energy efficiency linearly from 100%, the speed is recovered exponentially due to Boltzmanns law. Experimental data on a major biomotor (kinesin) suggest that the regime of universal optimization has been largely approached in living cells, underpinning the extreme efficiency-speed trade-off in biomotors. The universal optimization and its practical approachability are unique thermodynamic advantages of molecular systems over macroscopic engines in facilitating motor functions. The findings have important implications for the natural evolution of biomotors as well as the development of artificial counterparts.

Transcriptional Repressor TrmBL2 from Thermococcus kodakarensis Forms Filamentous Nucleoprotein Structures and Competes with Histones for DNA Binding in a Salt- and DNA Supercoiling-dependent Manner

Background: TrmBL2 and histones maintain the genome functioning in hyperthermophilic euryarchaeal cells. Results: TrmBL2 forms filaments on DNA through cooperative binding and competes with histones in a salt- and DNA supercoiling-dependent manner. Conclusion: TrmBL2-histone collective behavior dynamically controls DNA organization. Significance: The discovered mechanisms provide insights into the regulation of the genome structure and global transcription profile by TrmBL2 and histones. Architectural DNA proteins play important roles in the chromosomal DNA organization and global gene regulation in living cells. However, physiological functions of some DNA-binding proteins from archaea remain unclear. Recently, several abundant DNA-architectural proteins including histones, Alba, and TrmBL2 have been identified in model euryarchaeon Thermococcus kodakarensis. Although histones and Alba proteins have been previously characterized, the DNA binding properties of TrmBL2 and its interplay with the other major architectural proteins in the chromosomal DNA organization and gene transcription regulation remain largely unexplored. Here, we report single-DNA studies showing that at low ionic strength (<300 mm KCl), TrmBL2 binds to DNA largely in non-sequence-specific manner with positive cooperativity, resulting in formation of stiff nucleoprotein filamentous patches, whereas at high ionic strength (>300 mm KCl) TrmBL2 switches to more sequence-specific interaction, suggesting the presence of high affinity TrmBL2-filament nucleation sites. Furthermore, in vitro assays indicate the existence of DNA binding competition between TrmBL2 and archaeal histones B from T. kodakarensis, which can be strongly modulated by DNA supercoiling and ionic strength of surrounding solution. Overall, these results advance our understanding of TrmBL2 DNA binding properties and provide important insights into potential functions of architectural proteins in nucleoid organization and gene regulation in T. kodakarensis.

Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies

Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions of DNA and proteins. An important aspect of such studies is to obtain the dynamics of interactions; however the initial binding is often difficult to obtain due to large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid solution exchange method for magnetic tweezers single-molecule manipulation experiments, which is achieved by tethering the molecules inside microwells (typical dimensions–diameter (D): 40–50 μm, height (H): 100 μm; H:D∼2:1). Our simulations and experiments show that the flow speed can be reduced by several orders of magnitude near the bottom of the microwells from that in the flow chamber, effectively eliminating the flow disturbance to molecules tethered in the microwells. We demonstrate a wide scope of applications of this method by measuring the force dependent DNA structural transitions in response to solution condition change, and polymerization dynamics of RecA on ssDNA/SSB-coated ssDNA/dsDNA of various tether lengths under constant forces, as well as the dynamics of vinculin binding to α-catenin at a constant force (< 5 pN) applied to the α-catenin protein.

mDia1 senses both force and torque during F-actin filament polymerization

Formins, an important family of force-bearing actin-polymerizing factors, function as homodimers that bind with the barbed end of actin filaments through a ring-like structure assembled from dimerized FH2 domains. It has been hypothesized that force applied to formin may facilitate transition of the FH2 ring from an inhibitory closed conformation to a permissive open conformation, speeding up actin polymerization. We confirm this hypothesis for mDia1 dependent actin polymerization by stretching a single-actin filament in the absence of profilin using magnetic tweezers, and observe that increasing force from 0.5 to 10 pN can drastically speed up the actin polymerization rate. Further, we find that this force-promoted actin polymerization requires torsionally unconstrained actin filament, suggesting that mDia1 also senses torque. As actin filaments are subject to complex mechanical constraints in living cells, these results provide important insights into how formin senses these mechanical constraints and regulates actin organization accordingly.Formins are actin-polymerisation factors that are sensitive to force. Here the authors find that pulling force on an actin filament promoted faster actin polymerisation by the formin mDia1, and also found that the actin filament must be torsionally unconstrained, suggesting that mDia1 can also sense torque.

Transfer-matrix calculations of the effects of tension and torque constraints on DNA–protein interactions

Abstract Organization and maintenance of the chromosomal DNA in living cells strongly depends on the DNA interactions with a plethora of DNA-binding proteins. Single-molecule studies show that formation of nucleoprotein complexes on DNA by such proteins is frequently subject to force and torque constraints applied to the DNA. Although the existing experimental techniques allow to exert these type of mechanical constraints on individual DNA biopolymers, their exact effects in regulation of DNA–protein interactions are still not completely understood due to the lack of systematic theoretical methods able to efficiently interpret complex experimental observations. To fill this gap, we have developed a general theoretical framework based on the transfer-matrix calculations that can be used to accurately describe behaviour of DNA–protein interactions under force and torque constraints. Potential applications of the constructed theoretical approach are demonstrated by predicting how these constraints affect the DNA-binding properties of different types of architectural proteins. Obtained results provide important insights into potential physiological functions of mechanical forces in the chromosomal DNA organization by architectural proteins as well as into single-DNA manipulation studies of DNA–protein interactions.

Force dependence of filopodia adhesion: involvement of myosin II and formins

Filopodia are dynamic membrane protrusions driven by polymerization of an actin filament core, mediated by formin molecules at the filopodia tips. Filopodia can adhere to the extracellular matrix and experience both external and cell generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we induced sustained growth of filopodia by applying pulling force to their tips via attached fibronectin-coated beads trapped by optical tweezers. Strikingly, pharmacological inhibition or knockdown of myosin IIA, which localized to the base of filopodia, resulted in weakening of filopodia adherence strength. Inhibition of formins, which caused detachment of actin filaments from formin molecules, produced similar effect. Thus, myosin IIA-generated centripetal force transmitted to the filopodia tips through interactions between formins and actin filaments is required for filopodia adhesion. Force-dependent adhesion led to preferential attachment of filopodia to rigid versus fluid substrates, which may underlie cell orientation and polarization.

Theoretical Methods for Studying DNA Structural Transitions under Applied Mechanical Constraints

Recent progress in single-molecule manipulation technologies has made it possible to exert force and torque on individual DNA biopolymers to probe their mechanical stability and interaction with various DNA-binding proteins. It was revealed in these experiments that the DNA structure and formation of nucleoprotein complexes by DNA-architectural proteins can be strongly modulated by an intricate interplay between the entropic elasticity of DNA and its global topology, which is closely related to the mechanical constraints applied to the DNA. Detailed understanding of the physical processes underlying the DNA behavior observed in single-molecule experiments requires the development of a general theoretical framework, which turned out to be a rather challenging task. Here, we review recent advances in theoretical methods that can be used to interpret single-molecule manipulation experiments on DNA.

Effects of Mechanical Stimuli on Profilin- and Formin-Mediated Actin Polymerization

Self-assembling actin filaments not only form the basis of the cytoskeleton network in cells but also are utilized as nanosized building blocks to make novel active matter in which the dynamic polymerization and depolymerization of actin filaments play a key role. Formins belong to a main family of actin nucleation factors that bind to the barbed end of actin filaments and regulate actin polymerization through an interaction with profilin. Due to actomyosin contractility and relative rotation between formin and actin filaments, formin-dependent actin polymerization is subject to force and rotation constraints. However, it remains unclear how force and rotation constraints affect formin-dependent actin polymerization in the presence of profilin. Here, we show that for rotation-unconstrained actin filaments, elongation is accelerated by both force and profilin. The combined effect leads to surprisingly fast actin elongation that can approach the diffusion-limited rate at forces of a few piconewtons. The elongation of rotation-constrained filaments is also accelerated by profilin but is insensitive to applied force. We show that FH2, the main actin binding domain, plays the primary mechanosensing role. Together, the findings not only significantly advance our understanding of the mechanochemical regulation of formin-mediated actin polymerization in cells but also can potentially be utilized to make novel actin-based active matter.

Atomic-Scale Insights into Physical Mechanisms Driving Enzymes’ “Working Cycles”

Artem K. Efremov1,2,* and Fazoil I. Ataullakhanov3,4,5,6,* Mechanobiology Institute, Centre for Bioimaging Sciences, National University of Singapore, Singapore, Singapore; Center for Theoretical Problems of Physico-Chemical Pharmacology, Russian Academy of Sciences, Moscow, Russia; Dmitry Rogachev National Research Center for Hematology, Oncology, and Immunology, Moscow, Russia; Moscow State University, Moscow, Russia; and Moscow Institute of Physics and Technology, Moscow, Russia