Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, binds Toll-like receptor 4 (TLR4)-MD2 complex and activates innate immune responses. LPS transfer to ...TLR4-MD2 is catalyzed by both LPS binding protein (LBP) and CD14. To define the sequential molecular interactions underlying this transfer, we reconstituted in vitro the entire LPS transfer process from LPS micelles to TLR4-MD2. Using electron microscopy and single-molecule approaches, we characterized the dynamic intermediate complexes for LPS transfer: LBP-LPS micelles, CD14-LBP-LPS micelle, and CD14-LPS-TLR4-MD2 complex. A single LBP molecule bound longitudinally to LPS micelles catalyzed multi-rounds of LPS transfer to CD14s that rapidly dissociated from LPB-LPS complex upon LPS transfer via electrostatic interactions. Subsequently, the single LPS molecule bound to CD14 was transferred to TLR4-MD2 in a TLR4-dependent manner. The definition of the structural determinants of the LPS transfer cascade to TLR4 may enable the development of targeted therapeutics for intervention in LPS-induced sepsis.
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•Observation of dynamic intermediated formed during LPS transfer by TEM•Reconstitution of the entire LPS transfer cascade to TLR4-MD2 on TIRF microscopy•Identification of key charged residues in LBP and CD14 for dynamic LPS transfer•CD14-LPS complex interact with TLR4 LRR13-LRR15 domain for LPS transfer to MD2
Lipopolysaccharide (LPS) of Gram-negative bacteria activates host innate immune responses. Ryu et al. investigates dynamic intermediates in the LBP-CD14-mediated LPS transfer to TLR4-MD2 down to the single-molecule resolution with negative-stain TEM and TIRF analysis, thus identifying the key molecular determinants in LBP, CD14, and TLR4 for efficient LPS transfer.
To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical ...membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the
rhomboid protease GlpG and the human β
-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding.
This chapter explores advanced single-molecule techniques for studying protein-DNA interactions, particularly focusing on Replication Protein A (RPA) using a force-fluorescence setup. It combines ...magnetic tweezers (MT) with total internal reflection fluorescence (TIRF) microscopy, enabling detailed observation of DNA behavior under mechanical stress. The chapter details the use of DNA hairpins and bare DNA to examine RPA's binding dynamics and its influence on DNA's mechanical properties. This approach provides deeper insights into RPA's role in DNA replication, repair, and recombination, highlighting its significance in maintaining genomic stability.
Mechanical forces play pivotal roles in regulating cell shape, function, and fate. Key players that govern the mechanobiological interplay are the mechanosensitive proteins found on cell membranes ...and in cytoskeleton. Their unique nanomechanics can be interrogated using single-molecule tweezers, which can apply controlled forces to the proteins and simultaneously measure the ensuing structural changes. Breakthroughs in high-resolution tweezers have enabled the routine monitoring of nanometer-scale, millisecond dynamics as a function of force. Undoubtedly, the advancement of structural biology will be further fueled by integrating static atomic-resolution structures and their dynamic changes and interactions observed with the force application techniques. In this minireview, we will introduce the general principles of single-molecule tweezers and their recent applications to the studies of force-bearing proteins, including the synaptic proteins that need to be categorized as mechanosensitive in a broad sense. We anticipate that the impact of nano-precision approaches in mechanobiology research will continue to grow in the future.
During intracellular membrane trafficking, N-ethylmaleimide-sensitive factor (NSF) and alpha-soluble NSF attachment protein (α-SNAP) disassemble the soluble NSF attachment protein receptor (SNARE) ...complex for recycling of the SNARE proteins. The molecular mechanism by which NSF disassembles the SNARE complex is largely unknown. Using single-molecule fluorescence spectroscopy and magnetic tweezers, we found that NSF disassembled a single SNARE complex in only one round of adenosine triphosphate (ATP) turnover. Upon ATP cleavage, the NSF hexamer developed internal tension with dissociation of phosphate ions. After latent time measuring tens of seconds, NSF released the built-up tension in a burst within 20 milliseconds, resulting in disassembly followed by immediate release of the SNARE proteins. Thus, NSF appears to use a "spring-loaded" mechanism to couple ATP hydrolysis and unfolding of substrate proteins.
Abstract
The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large ...conformational changes within the ∼50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20–40 nm at forces of 1.0–0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ∼200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.
Submicrometer elasticity of double-stranded DNA (dsDNA) governs nanoscale bending of DNA segments and their interactions with proteins. Single-molecule force spectroscopy, including magnetic tweezers ...(MTs), is an important tool for studying DNA mechanics. However, its application to short DNAs under 1 μm is limited. We developed an MT-based method for precise force-extension measurements in the 100-nm regime that enables in situ correction of the error in DNA extension measurement, and normalizes the force variability across beads by exploiting DNA hairpins. The method reduces the lower limit of tractable dsDNA length down to 198 base pairs (bp) (67 nm), an order-of-magnitude improvement compared to conventional tweezing experiments. Applying this method and the finite worm-like chain model we observed an essentially constant persistence length across the chain lengths studied (198 bp to 10 kbp), which steeply depended on GC content and methylation. This finding suggests a potential sequence-dependent mechanism for short-DNA elasticity.
Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of ...mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.
Mechanical forces play pivotal roles in regulating cell shape, function, and fate. Key players that govern the mechanobiological interplay are the mechanosensitive proteins found on cell membranes ...and in cytoskeleton. Their unique nanomechanics can be interrogated using single-molecule tweezers, which can apply controlled forces to the proteins and simultaneously measure the ensuing structural changes. Breakthroughs in high-resolution tweezers have enabled the routine monitoring of nanometer-scale, millisecond dynamics as a function of force. Undoubtedly, the advancement of structural biology will be further fueled by integrating static atomic-resolution structures and their dynamic changes and interactions observed with the force application techniques. In this minireview, we will introduce the general principles of single-molecule tweezers and their recent applications to the studies of force-bearing proteins, including the synaptic proteins that need to be categorized as mechanosensitive in a broad sense. We anticipate that the impact of nano-precision approaches in mechanobiology research will continue to grow in the future.
•Rheotaxic movement of zebrafish larvaecan characterize biological impacts of drug candidates.•A hardware-software-combined automated platform was developed for aquatic behavior analysis.•A ...combination of head angle and velocity could track changes in catch-up behaviors of rheotaxis.•Rheotactic characteristics can be efficient biomarkers for drug screening purposes.
Aquatic animals have rheotaxis that maintains a balance in response to water flow.They sense water flow through hair cells in lateral line, thereby leading to behavior changes relevant to damages on hair cells, the primary sensory receptor cells within auditory and vestibular systems.Zebrafish are efficient animal models for high-throughput drug screening with human-like hair cells along the lateral line. Their rheotactic behaviors could be assays for hair-cell-targeted drug screening. However,knowledge and tools for rheotaxis analysis along the extent of hair-celldamage have not beenfullyinvestigated. Thisarticleaims atcharacterization of rheotactic behaviorsidentifyinglateral linestatesviaananalysis platformthatsimultaneously examinesmultiple zebrafish larvae.To this end, we developed anautomated framework that incorporated animal test hardware equipment and real-time analysis software for monitoring aquatic behaviors of multiple larvae. Through this framework, a commensurable measure for one-dimensional characterization of rheotactic behaviors was consolidated so that its linear changes could be associated with the population of hair cells remaining intact. Thesefindingssatisfied requestsfor an automated analysis platform to conduct large-scale screening anda biomarkerthatdiscriminatethe seriousness of haircell damagetoscreen candidates having significant effectsinotoprotectivedrug discovery.
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