Single-molecule imaging reveals a direct role of CTCF's zinc fingers in SA interaction and cluster-dependent RNA recruitment.

CTCF is a zinc finger protein associated with transcription regulation that also acts as a barrier factor for topologically associated domains (TADs) generated by cohesin via loop extrusion. These processes require different properties of CTCF-DNA interaction, and it is still unclear how CTCF's structural features may modulate its diverse roles. Here, we employ single-molecule imaging to study both full-length CTCF and truncation mutants. We show that CTCF enriches at CTCF binding sites (CBSs), displaying a longer lifetime than observed previously. We demonstrate that the zinc finger domains mediate CTCF clustering and that clustering enables RNA recruitment, possibly creating a scaffold for interaction with RNA-binding proteins like cohesin's subunit SA. We further reveal a direct recruitment and an increase of SA residence time by CTCF bound at CBSs, suggesting that CTCF-SA interactions are crucial for cohesin stability on chromatin at TAD borders. Furthermore, we establish a single-molecule T7 transcription assay and show that although a transcribing polymerase can remove CTCF from CBSs, transcription is impaired. Our study shows that context-dependent nucleic acid binding determines the multifaceted CTCF roles in genome organization and transcription regulation.

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

DNA curtains for studying phase separation mechanisms of DNA-organizing proteins.

Phase separation is one key mechanism to organize chromatin into compartments and to regulate the activity of the genome. The formation of liquid-like droplets within the nucleus is driven by protein association to the DNA via multivalent binding and the recruitment of other proteins building a concentrated reaction environment. Common methods to study phase separation and its liquid-like nature are based on microscopy of the formed droplets but lack the resolution to obtain information on the molecular level. Here, we describe the application of the DNA curtain technique for studying protein-mediated phase separation on DNA. For this, multiple lipid-anchored DNA strands are flow-stretched across a nanobarrier to allow single-molecule studies of protein-DNA interactions in a high-throughput approach. Our protocol describes how protein-induced DNA compaction can be observed in real-time and which wash protocols are suitable to characterize the interactions that promote condensate formation. Furthermore, we demonstrate how fluorescently labeled tracer proteins can serve as orientation points to examine the DNA compaction mechanism in detail.

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. We investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner qualitatively agree with our data. We speculate that 'monkey-bar' mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Single-molecule experiments reveal the elbow as an essential folding guide in SMC coiled-coil arms.

Structural maintenance of chromosome (SMC) complexes form ring-like structures through exceptional elongated coiled-coils (CCs). Recent studies found that variable CC conformations, including open and collapsed forms, which might result from discontinuities in the CC, facilitate the diverse functions of SMCs in DNA organization. However, a detailed description of the SMC CC architecture is still missing. Here, we study the structural composition and mechanical properties of SMC proteins with optical tweezers unfolding experiments using the isolated Psm3 CC as a model system. We find a comparatively unstable protein with three unzipping intermediates, which we could directly assign to CC features by crosslinking experiments and state-of-the-art prediction software. Particularly, the CC elbow is shown to be a flexible, potentially non-structured feature, which divides the CC into sections, induces a pairing shift from one CC strand to the other and could facilitate large-scale conformational changes, most likely via thermal fluctuations of the flanking CC sections. A replacement of the elbow amino acids hinders folding of the consecutive CC region and frequently leads to non-native misalignments, revealing the elbow as a guide for proper folding. Additional in vivo manipulation of the elbow flexibility resulted in impaired cohesin complexes, which directly link the sensitive CC architecture to the biological function of cohesin.

Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix.

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs), superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between partially folded and unfolded conformations. We rationalize the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the center toward both termini simultaneously. Most strikingly, we also directly observe the protein's superhelical tertiary structure in the force signal. Using protein engineering, crystallography, and single-molecule experiments, we show that the superhelical geometry can be altered by carefully placed amino acid substitutions, and we examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications.

Identification and correction of miscalibration artifacts based on force noise for optical tweezers experiments.

Single-molecule force spectroscopy using optical tweezers continues to provide detailed insights into the behavior of nanoscale systems. Obtaining precise measurements of their mechanical properties is highly dependent on accurate instrument calibration. Therefore, instrumental drift or inaccurate calibration may prevent reaching an accuracy at the theoretical limit and may lead to incorrect conclusions. Commonly encountered sources of error include inaccuracies in the detector sensitivity and trap stiffness and neglecting the non-harmonicity of an optical trap at higher forces. Here, we first quantify the impact of these artifacts on force-extension data and find that a small deviation of the calibration parameters can already have a significant downstream effect. We then develop a method to identify and remove said artifacts based on differences in the theoretical and measured noise of bead fluctuations. By applying our procedure to both simulated and experimental data, we can show how effects due to miscalibration and trap non-linearities can be successfully removed. Most importantly, this correction can be performed post-measurement and could be adapted for data acquired using any force spectroscopy technique.

Slow Transition Path Times Reveal a Complex Folding Barrier in a Designed Protein.

De-novo designed proteins have received wide interest as potential platforms for nano-engineering and biomedicine. While much work is being done in the design of thermodynamically stable proteins, the folding process of artificially designed proteins is not well-studied. Here we used single-molecule force spectroscopy by optical tweezers to study the folding of ROSS, a de-novo designed 2x2 Rossmann fold. We measured a barrier crossing time in the millisecond range, much slower than what has been reported for other systems. While long transition times can be explained by barrier roughness or slow diffusion, we show that isotropic roughness cannot explain the measured transition path time distribution. Instead, this study shows that the slow barrier crossing of ROSS is caused by the population of three short-lived high-energy intermediates. In addition, we identify incomplete and off-pathway folding events with different barrier crossing dynamics. Our results hint at the presence of a complex transition barrier that may be a common feature of many artificially designed proteins.

Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding.

Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. In the current work we investigate the influence of loop length variation on the free energies of folding and ligand binding in a small globular single-domain protein containing two EF-hand subdomains-calbindin D9k. We introduce a linker extension between the subdomains and vary its length between 1 to 16 glycine residues. We find a close to linear relationship between the linker length and the free energy of folding of the Ca2+-free protein. In contrast, the linker length has only a marginal effect on the Ca2+ affinity and cooperativity. The variant with a single-glycine extension displays slightly increased Ca2+ affinity, suggesting that the slightly extended linker allows optimized packing of the Ca2+-bound state. For the extreme case of disconnected subdomains, Ca2+ binding becomes coupled to folding and assembly. Still, a high affinity between the EF-hands causes the non-covalent pair to retain a relatively high apparent Ca2+ affinity. Our results imply that loop length variation could be an evolutionary option for modulating properties such as protein stability and turnover without compromising the energetics of the specific function of the protein.

A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules.

Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a "permanent bridge" resisting forces over 80 pN and a force-sensitive "reversible bridge." The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. "Permanent" cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.

A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy.

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183-383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

Single-Molecule Imaging Reveals a Collapsed Conformational State for DNA-Bound Cohesin.

Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here, we use single-molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. Furthermore, we demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin.

Ligand-induced changes of the apparent transition-state position in mechanical protein unfolding.

Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx(‡) of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx(‡) can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx(‡), depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.

Sequence-resolved free energy profiles of stress-bearing vimentin intermediate filaments.

Intermediate filaments (IFs) are key to the mechanical strength of metazoan cells. Their basic building blocks are dimeric coiled coils mediating hierarchical assembly of the full-length filaments. Here we use single-molecule force spectroscopy by optical tweezers to assess the folding and stability of coil 2B of the model IF protein vimentin. The coiled coil was unzipped from its N and C termini. When pulling from the C terminus, we observed that the coiled coil was resistant to force owing to the high stability of the C-terminal region. Pulling from the N terminus revealed that the N-terminal half is considerably less stable. The mechanical pulling assay is a unique tool to study and control seed formation and structure propagation of the coiled coil. We then used rigorous theory-based deconvolution for a model-free extraction of the energy landscape and local stability profiles. The data obtained from the two distinct pulling directions complement each other and reveal a tripartite stability of the coiled coil: a labile N-terminal half, followed by a medium stability section and a highly stable region at the far C-terminal end. The different stability regions provide important insight into the mechanics of IF assembly.

Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy.

In this study we expand the accessible dynamic range of single-molecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 µs thus approaching the timescales accessible for all atom molecular dynamics simulations.

Dynamic force sensing of filamin revealed in single-molecule experiments.

Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20-21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that filamin force sensing is a highly dynamic process occurring in rapid equilibrium that increases the affinity to the target peptides by up to a factor of 17 between 2 and 5 pN. The equilibrium mechanism we find here can offer a general scheme for cellular force sensing.

Calcium-dependent folding of single calmodulin molecules.

Calmodulin is the primary calcium binding protein in living cells. Its function and structure depend strongly on calcium concentration. We used single molecule force spectroscopy by optical tweezers to study the folding of calmodulin in the physiologically relevant range. We find that full-length calmodulin switches from a rich and complex folding behavior at high calcium to a simple folding pathway at apo conditions. Using truncation mutants, we studied the individual domains separately. Folding and stability of the individual domains differ significantly at low calcium concentrations. With increasing calcium, the folding rate constants increase while unfolding rate constants decrease. The complete kinetic as well as energetic behavior of both domains could be modeled using a calcium-dependent three-pathway model. We find that the dominant folding pathway at high calcium concentrations proceeds via a transition state capable of binding one calcium ion. The folding of calmodulin seems to be designed to occur fast robustly over a large range of calcium concentrations and hence energetic stabilities.

Hidden Markov analysis of trajectories in single-molecule experiments and the effects of missed events.

The ever more complex fluctuation patterns discovered by single molecule experiments require statistical methods to analyze multi-state hopping traces of long lengths. Hidden Markov modeling is a statistical tool that offers the scalability to analyze even complex data and extract kinetic information. We give an introduction on how to implement hidden Markov modeling for the analysis of single molecule force spectroscopic traces, deal with missed events, and test the method on a calcium binding protein.

The complex folding network of single calmodulin molecules.

Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.