Biology subjects

Stigler, J.

Publications and source records attributed to Stigler, J..

8 recordsLinked to original sources

bioRxiv2026

Intra-Protein Interfaces Control Folding Dynamics and Mechanical Stability in a De Novo Designed Repeat Protein

Protein design has made it possible to generate stable structures with high accuracy, but the principles that determine how designed proteins fold and respond to force remain less understood. Repeat proteins provide a powerful test case because their stability is distributed across modular structural units and nearest-neighbor interfaces rather than concentrated in a single hydrophobic core. Here, we used single-molecule optical tweezers, intramolecular crosslinking and molecular dynamics simulations to dissect the folding landscape of DHR14R, a de novo designed helical repeat protein with a deep-learning-redesigned sequence. By introducing site-specific intramolecular crosslinks, we imposed defined boundary conditions on the repeat array and directly manipulated its mechanical folding pathway. DHR14R unfolds reversibly from its mechanically weakest boundary, the N-terminus, through a series of discrete intermediates. Refolding begins with the formation of a three-helix seed rather than a complete two-helix repeat, showing that the structural repeat is not the elementary cooperative folding unit. Preformed terminal interfaces accelerate folding, indicating that seed formation is rate-limiting. Stabilizing both termini eliminates the low-force terminal-fraying pathway and forces disruption of internal interfaces, increasing mechanical stability more than twofold. Together, these results show that designed repeat-protein folding is governed by seed formation, interface propagation, and terminal boundary conditions. They establish intramolecular crosslinking as a strategy for rationally reshaping folding landscapes in designed proteins.

biophysics↗

bioRxiv2025

Single-Molecule Imaging Reveals DNA Shape Read-out by the INO80 Chromatin Remodeler

ATP-dependent chromatin remodeling enzymes act together to construct complex chromatin architectures around functionally important regulatory regions like promoters and origins of replication. The INO80 complex accurately positions nucleosomes bordering these regions, which is crucial for proper transcription start site selection and efficient replication. How INO80 localizes these nucleosomes and regulates its remodeling activity to produce specific positions mechanistically is unclear, although recent findings suggest a role for DNA shape features and barrier factors. Here, we use single-molecule DNA curtains to directly observe interactions of INO80 with DNA, nucleosomes and barrier factor Reb1. We show that INO80 recognizes DNA shape and strongly binds to shape features that are enriched in promoters and ORIs. Moreover, we find that INO80 performs 1D searches and intersegmental transfers to dynamically interact with nucleosomes and Reb1, but cannot bypass them, indicating that they could confine INO80 within promoters and thereby increase engagement with flanking nucleosomes. Our findings reveal how INO80s target-site search and remodeling activity are influenced by DNA shape recognition and molecular architecture and elucidate how remodelers can integrate different kinds of information to transform the chromatin landscape.

biochemistry↗

bioRxiv2024

LINE-1 ribonucleoprotein complexes bind DNA to enable nuclear entry during mitosis

Long Interspersed Nuclear Element-1 (LINE-1) is an autonomous retrotransposon that makes up a significant portion of the human genome, contributing to genetic diversity and genome evolution. LINE-1 encodes two proteins, ORF1p and ORF2p, both essential for successful retrotransposition. ORF2p has endonuclease and reverse transcription activity, while ORF1p binds RNA. Many copies of ORF1p assemble onto the LINE-1 RNA to form a ribonucleoprotein complex (RNP). However, the functional role of RNPs in the LINE-1 life cycle is unclear. Using reconstitution assays on DNA curtains, we show that L1 RNPs gain DNA binding activity only when RNA is super-saturated with ORF1p. In cells, L1 RNPs bind to chromosomes during mitosis. Mutational analysis reveals that DNA binding is crucial for nuclear entry and LINE-1 retrotransposition activity. Thus, a key function of ORF1p is to form an RNP that gains access to the genome through DNA binding upon nuclear envelope breakdown.

biophysics↗

bioRxiv2023

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 CTCFs 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 cohesins 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 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.

molecular biology↗

bioRxiv2023

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 ISWIs mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWIs 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.

biochemistry↗

bioRxiv2021

The loader complex Scc2/4 forms co-condensates with DNA as loading sites for cohesin

The genome is organized by diverse packaging mechanisms like nucleosome formation, loop extrusion and phase separation, which all compact DNA in a dynamic manner. Phase separation additionally drives protein recruitment to condensed DNA sites and thus regulates gene transcription. The cohesin complex is a key player in chromosomal organization that extrudes loops to connect distant regions of the genome and ensures sister chromatid cohesion after S-phase. For stable loading onto the DNA and for activation, cohesin requires the loading complex Scc2/4. As the precise loading mechanism remains unclear, we investigated whether phase separation might be the initializer of the cohesin recruitment process. We found that, in absence of cohesin, budding yeast Scc2/4 forms phase separated co-condensates with DNA, which comprise liquid-like properties shown by droplet shape, fusion ability and reversibility. We reveal in DNA curtain and optical tweezer experiments that these condensates are built by DNA bridging and bending through Scc2/4. Importantly, Scc2/4-mediated condensates recruit cohesin efficiently and increase the stability of the cohesin complex. We conclude that phase separation properties of Scc2/4 enhance cohesin loading by molecular crowding, which might then provide a starting point for the recruitment of additional factors and proteins.

biophysics↗

bioRxiv2021

Consensus tandem tetratricopeptide repeat proteins are complex superhelical nanosprings

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 folded and unfolded conformations. We rationalise the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the centre towards both termini simultaneously. Strikingly, we also directly observe the proteins superhelical tertiary structure in the force signal. Using protein engineering, crystallography and single-molecule experiments, we show how the superhelical geometry can be altered by carefully placed amino-acid substitutions and 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. Significance statementRepetition of biological building blocks is crucial to modulating and diversifying structure and function of biomolecules across all organisms. In tandem-repeat proteins, the linear arrangement of small structural motifs leads to the formation of striking supramolecular shapes. Using a combination of single-molecule biophysical techniques and modelling approaches, we dissect the spring-like nature of a designed repeat protein and demonstrate how its shape and mechanics can be manipulated by design. These novel insights into the biomechanical and biochemical characteristics of this protein class give us a methodological basis from which to understand the biological functions of repeat proteins and to exploit them in nanotechnology and biomedicine.

biophysics↗

bioRxiv2019

A conserved activity for cohesin in bridging DNA molecules

Essential processes such as accurate chromosome segregation, regulation of gene expression and DNA repair rely on protein-mediated DNA tethering. Sister chromatid cohesion requires the SMC complex cohesin to act as a protein linker that holds replicated chromatids together (1, 2). The molecular mechanism by which cohesins hold sister chromatids has remained controversial. Here, we used a single molecule approach to visualise the activity of cohesin complexes as they hold DNA molecules. We describe a DNA bridging activity that requires ATP and is conserved from yeast to human cohesin. We show that cohesin can form two distinct classes of bridges at physiological conditions, a \"permanent bridge\" able to resists high force (over 80pN) and a \"reversible bridge\" that breaks at lower forces (5-40pN). Both classes of bridges require Scc2/Scc4 in addition to ATP. We demonstrate that bridge formation requires physical proximity of the DNA segments to be tethered and show that \"permanent\" cohesin bridges can move between two DNA molecules but cannot be removed from DNA when they occur in cis. This suggests that separate physical compartments in cohesin molecules are involved in the bridge. Finally, we show that cohesin tetramers, unlike condensin, cannot compact linear DNA molecules against low force, demonstrating that the core activity of cohesin tetramers is bridging DNA rather than compacting it. Our findings carry important implications for the understanding of the basic mechanisms behind cohesin-dependent establishment of sister chromatid cohesion and chromosome architecture.

biochemistry↗