Biology subjects

Marklund, E.

Publications and source records attributed to Marklund, E..

10 recordsLinked to original sources

bioRxiv2026

Native mass spectrometry of membrane protein lipid and ligand complexes using de novo designed WRAP domains

Membrane proteins engage in dynamic transient interactions with surrounding lipids along with their substrates. Capturing these concomitant contacts often remains a challenging problem. Here, we demonstrate that generative deep learning-designed WRAP domains can preserve weak interactions of membrane protein complexes in native mass spectrometry. Using WRAP-fused GlpG, AqpM, and OmpA as model systems, we show that lipid interactions can be retained and characterized without detergent micelles. We use the approach to show that WRAP-OmpA selectively binds phosphatidylethanolamine lipids within cavities formed at the protein-WRAP interface, while simultaneously accommodating weak chitobiose ligand binding. These findings establish WRAPs as versatile vehicles to probe ligand and lipid interactions of membrane proteins.

biophysics↗

bioRxiv2025

The lac repressor strikes a balance between inducibility and strength of repression

Transcription factors (TFs) efficiently locate their target DNA sequences by combining three-dimensional diffusion and one-dimensional sliding on nonspecific DNA. To balance rapid sliding with strong specific binding, TFs were proposed to switch between search and recognition conformations. For E. coli lac repressor (LacI), the folding of the hinge helices has been implicated in the conformational switch. Here, we tested how mutations in the hinge region impact the search speed and binding stability. Based on molecular dynamics simulations, we selected two LacI mutants favoring either search or recognition conformation. We measured the binding kinetics of the mutants both in vitro on DNA microarrays with 2,479 different Lac operators and in vivo via single-molecule experiments. We conclude that a hinge region mutation causing less helix propensity enhances the specificity but reduces binding strength globally, while a hinge region mutation causing higher helix propensity has opposite effects. However, altered specificity impacts the search time less than expected. Instead, the major effect was impaired dissociation in response to IPTG induction for the strongly binding mutant. Together with earlier reports of affinity-inducibility trade-offs in LacI, our data support the model in which the hinge region governs a trade-off between binding stability and inducibility rather than between speed and binding stability.

molecular biology↗

bioRxiv2025

Thermodynamic principles link in vitro transcription factor affinities to single-molecule chromatin states in cells

The molecular details governing transcription factor (TF) binding and the formation of accessible chromatin are not yet quantitatively understood - including how sequence context modulates affinity, how TFs search DNA, the kinetics of TF occupancy, and how motif grammars coordinate binding. To resolve these questions for a human TF, erythroid Kruppel-like factor (eKLF/KLF1), we quantitatively compare, in high throughput, in vitro TF binding rates and affinities with in vivo single molecule TF and nucleosome occupancies across engineered DNA sequences. We find that 40-fold flanking sequence effects on affinity are consistent with distal flanks tuning TF search parameters and captured by a linear energy model. Motif recognition probability, rather than time in the bound state, drives affinity changes, and in vitro and in nuclei measurements exhibit consistent, minutes-long TF residence times. Finally, pairing in vitro biophysical parameters with thermodynamic models accurately predicts in vivo single-molecule chromatin states for unseen motif grammars. HighlightsO_LIKLF1:DNA binding is consistent with a three state binding model wherein the probability of recognizing the motif from a nonspecifically-bound state drives affinity. C_LIO_LISubstantial effects from proximal and distal motif-flanking sequence on KLF1 binding affinity are captured by an extended PWM model. C_LIO_LIKLF minutes-long residence time inferred from single-molecule footprinting in nuclei is consistent with in vitro measurement. C_LIO_LIIn vitro binding energies combined with thermodynamic models predict in vivo, single-molecule chromatin configurations across motif grammars and sequence contexts. C_LI

biophysics↗

bioRxiv2024

Single-molecule chromatin configurations link transcription factor binding to expression in human cells

The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TFs capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. This work provides a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration.

molecular biology↗

bioRxiv2024

High-Throughput DNA melt measurements enable improved models of DNA folding thermodynamics

DNA folding thermodynamics are central to many biological processes and biotechnological applications involving base-pairing. Current methods for predicting stability from DNA sequence use nearest-neighbor models that struggle to accurately capture the diverse sequence-dependency of elements other than Watson-Crick base pairs, likely due to insufficient experimental data. We introduce a massively parallel method, Array Melt, that uses fluorescence-based quenching signals to measure equilibrium stability of millions of DNA hairpins simultaneously on a repurposed Illumina sequencing flow cell. By leveraging this dataset of 27,732 sequences with two-state melting behavior, we derived a refined NUPACK-compatible nearest-neighbor model, a richer parameterization nearest-neighbor model that exhibits higher accuracy, and a graph neural network (GNN) model that identifies relevant interactions within DNA beyond nearest neighbors. All models provide improved accuracy in predicting DNA folding thermodynamics, providing improvements relevant for in silico design of qPCR primers, oligo hybridization probes, and DNA origami.

biophysics↗

bioRxiv2023

A 2-Hydroxybutyrate-mediated feedback loop regulates muscular fatigue

The metabolite 2-hydroxybutyrate (2HB) is produced by skeletal muscle acutely during exercise and persists for several hours in the blood post-exertion. We show here that 2HB directly inhibits branched- chain aminotransferase enzymes, and that this inhibition in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBP{beta} mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity both in vitro and in vivo, with the latter mimicking the effects of exercise training on whole body metabolism. Thus, we show here that 2-HB production by skeletal muscle represents a novel mechanism for the modification of metabolism by exercise.

physiology↗

bioRxiv2022

Short tandem repeats bind transcription factors to tune eukaryotic gene expression

Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and their polymorphisms alter gene expression, yet how they regulate transcription remains unknown. We find that STRs can modulate transcription factor (TF)-DNA affinities and on rates by up to 70-fold by directly binding TF DNA-binding domains, with energetic impacts approaching or exceeding mutations to consensus sites. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density near motifs to speed target search. Confirming that STRs also impact TF binding in cells, neural networks trained only on in vivo occupancies predict identical effects to those observed in vitro. Approximately 90% of TFs preferentially bind STRs that need not resemble known motifs, providing a novel cis-regulatory mechanism to target TFs to cognate sites.

biophysics↗

bioRxiv2021

Sequence specificity in DNA binding is determined by association rather than dissociation

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. Here, we derive a simple equation for target-site recognition, which uncovers a previously unrecognized coupling between the macroscopic association and dissociation rates of the searching protein. Importantly, this relationship makes it possible to recover the relevant microscopic rates from experimentally determined macroscopic ones. We directly test the equation by observing the binding and unbinding of individual lac repressor (LacI) molecules during target search. We find that LacI dissociates from different target sequences with essentially identical microscopic dissociation rates. Instead, sequence specificity is determined by the efficiency with which the protein recognizes different targets, effectively reducing its risk of being retained on a non-target sequence. Our theoretical framework also accounts for the coupling between off-target binding and unbinding of the catalytically inactive Cas9 (dCas9), showing that the binding pathway can be obtained from macroscopic data. One Sentence SummaryAssociation and dissociation rates are anti-correlated for reactions that include a nonspecific probing step.

biophysics↗

bioRxiv2020

Mechanism of DNA surface exploration and operator bypassing during target search

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 s. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

biophysics↗

bioRxiv2018

Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA1-5. It is however not known how sliding proteins are oriented with respect to DNA in order to recognize specific sequences. Here we measure the polarization of fluorescence emission from single fluorescently labeled lac repressor (LacI) molecules sliding on stretched DNA. Real-time feedback-coupled confocal single-particle tracking allows us to measure fluorescence correlation of the sliding molecules. We find that the fluctuations in the fluorescence signal on the s timescale are accurately described by rotation-coupled sliding on DNA. On average, LacI moves [~]50 base pairs per revolution, which is significantly longer than the 10.5 bp helical periodicity of DNA. Our data support a facilitated diffusion model1 where the transcription factor (TF) scans the DNA grooves for hydrogen bonding opportunities in a pre-aligned orientation with occasional slippage out of the groove.

biophysics↗