Biology subjects

Lipfert, J.

Publications and source records attributed to Lipfert, J..

31 records · Page 2Linked to original sources

bioRxiv2022

A DNA origami fiducial for accurate 3D AFM imaging

Atomic force microscopy (AFM) is a powerful technique for imaging molecules, macromolecular complexes, and nanoparticles with nanometer-resolution. However, AFM images are distorted by the shape of the tip used. These distortions can be corrected if the tip shape can be determined by scanning a sample with features sharper than the tip and higher than the object of interest. Here we present a 3D DNA origami structure as fiducial for tip reconstruction and image correction. Our fiducial is stable under a broad range of conditions and has sharp steps at different heights that enable reliable tip reconstruction from as few as ten fiducials. The DNA origami is readily co-deposited with biological and non-biological samples, achieves higher precision for the tip apex than polycrystalline samples, and dramatically improves the accuracy of the lateral dimensions determined from the images. Our fiducial thus enables accurate and precise AFM imaging for a broad range of applications.

biophysics↗

bioRxiv2021

DNA fluctuations reveal the size and dynamics of topological domains

DNA supercoiling is a key regulatory mechanism that orchestrates DNA readout, recombination, and genome maintenance. DNA-binding proteins often mediate these processes by bringing two distant DNA sites together, thereby inducing (transient) topological domains. In order to understand the dynamics and molecular architecture of protein induced topological domains in DNA, quantitative and time-resolved approaches are required. Here we present a methodology to determine the size and dynamics of topological domains in supercoiled DNA in real-time and at the single molecule level. Our approach is based on quantifying the extension fluctuations - in addition to the mean extension - of supercoiled DNA in magnetic tweezers. Using a combination of high-speed magnetic tweezers experiments, Monte Carlo simulations, and analytical theory, we map out the dependence of DNA extension fluctuations as a function of supercoiling density and external force. We find that in the plectonemic regime the extension variance increases linearly with increasing supercoiling density and show how this enables us to determine the formation and size of topological domains. In addition, we demonstrate how transient (partial) dissociation of DNA bridging proteins results in dynamic sampling of different topological states, which allows us to deduce the torsional stiffness of the plectonemic state and the kinetics of protein-plectoneme interactions. We expect our approach to enable quantification of the dynamics and reaction pathways of DNA processing enzymes and motor proteins, in the context of physiologically relevant forces and supercoiling densities. SignificanceIn the cell, long DNA molecules carry the genetic information and must be stored and maintained, yet remain accessible for read out and processing. DNA supercoiling facilitates compaction of DNA, modulates its accessibility, and spatially juxtaposes DNA sites distant in linear DNA sequence. By binding to two sites in supercoiled DNA, DNA bridging proteins can pinch off topological domains and alter DNA plectoneme dynamics. Here we show how DNA bridging and topological domain dynamics can be detected from changes in the extension fluctuations of supercoiled DNA molecules tethered in magnetic tweezers. Our work highlights how considering DNA extension fluctuations, in addition to the mean extension, provides additional information and enables the investigation of protein-DNA interactions that are otherwise invisible.

biophysics↗

bioRxiv2021

A Conformational Transition of Von Willebrand Factor's D'D3 Domain Primes It For Multimerization

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the (trans-)Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the DD3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose two previously buried cysteines that are known to be vital for multimerization. By characterizing the conformational change at varying levels of force, we are able to quantify the kinetics of the transition and the stability of the interface. We find a pronounced destabilization of the interface upon lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the DD3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII (FVIII), providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the DD3 domain in VWF biosynthesis and function and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

biophysics↗

bioRxiv2021

A Tethered Ligand Assay to Probe SARS-CoV-2:ACE2 Interactions

SARS-CoV-2 infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and multivalent interactions play critical roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2-5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate in comparison to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a novel way to investigate the roles of mutations and blocking agents for targeted pharmaceutical intervention.

biophysics↗

bioRxiv2021

Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

Nucleosomes are the basic units of chromatin and critical to the storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks of which post-translational modifications of histones play a key role. However, the mode of action and the structural implications on the single-molecule level of nucleosomes is often still poorly understood. Here, we apply a high-throughput AFM imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants H3K36me3, H3S10phos and H4K5/8/12/16ac. Our data set of >25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5 bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild type nucleosomes and additionally unwrap stochastically from both sides similar to CENP-A nucleosomes and in contrast to the highly anti-cooperative unwrapping of wild type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild type nucleosomes and also retain the same level of anti-cooperativity. Our findings help putting the mode of action of these modifications into context: While H3K36me3 likely partially acts by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes. Statement of SignificanceThe packing and readout of our genome is tightly regulated by post-translational histone modifications (PTMs). While a vast range of PTMs has been studied with respect to their implications for gene activity and replication, a detailed view of the direct effect of PTMs on conformational changes of nucleosomes is still lacking. Here we investigate the structural implications of several key modifications (H3K36me3, H3S10phos and H4K5/8/12/16ac) by high-throughput AFM imaging. Our findings enable a better understanding of the mode of action of these specific modifications and provide an analysis pipeline for the investigation of other epigenetic modifications.

molecular biology↗

bioRxiv2021

Twisting DNA by Salt

The structure and properties of DNA depend on the environment, in particular the ion atmosphere. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concentration and identity of the surrounding ions. To resolve how cations influence the helical twist, we combine single-molecule magnetic tweezer experiments and extensive all- atom molecular dynamics simulations. Two interconnected trends are observed for monovalent alkali and divalent alkaline earth cations. First, DNA twist increases monotonously with increasing concentration for all ions investigated. Second, for a given salt concentration, DNA twist strongly depends on cation identity. At 100 mM concentration, DNA twist increases as Ba2+ < Na+ < K+ < Rb+ < Li+ {approx} Cs+ < Sr2+ < Mg2+ < Ca2+. Our molecular dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the observed ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs+ and Sr2+. The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnology.

biophysics↗

bioRxiv2020

A Tethered Ligand Assay to Probe the SARS-CoV-2 ACE2 Interaction under Constant Force

The current COVID-19 pandemic has a devastating global impact and is caused by the SARS-CoV-2 virus. SARS-CoV-2 attaches to human host cells through interaction of its receptor binding domain (RBD) located on the viral Spike (S) glycoprotein with angiotensin converting enzyme-2 (ACE2) on the surface of host cells. RBD binding to ACE2 is a critical first step in SARS-CoV-2 infection. Viral attachment occurs in dynamic environments where forces act on the binding partners and multivalent interactions play central roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using specific molecular handles, we tether the fusion proteins between a functionalized flow cell surface and magnetic beads in magnetic tweezers. We observe repeated interactions of RBD and ACE2 under constant loads and can fully quantify the force dependence and kinetics of the binding interaction. Our results suggest that the SARS-CoV-2 ACE2 interaction has higher mechanical stability, a larger free energy of binding, and a lower off-rate than that of SARS-CoV-1, the causative agents of the 2002-2004 SARS outbreak. In the absence of force, the SARS-CoV-2 RBD rapidly (within [≤]1 ms) engages the ACE2 receptor if held in close proximity and remains bound to ACE2 for 400-800 s, much longer than what has been reported for other viruses engaging their cellular receptors. We anticipate that our assay will be a powerful tool investigate the roles of mutations in the RBD that might alter the infectivity of the virus and to test the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

biophysics↗

bioRxiv2020

Intercalative DNA binding governs fluorescence enhancement of SYBR Gold

SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its binding mode to DNA remains controversial. Here, we quantitate SYBR Gold binding to DNA using two complementary approaches. We use mechanical micromanipulation with magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length, twist, and mechanical properties. The MT assay reveals systematic lengthening and unwinding of DNA upon SYBR Gold binding, consistent with an intercalative binding mode where every SYBR Gold molecule unwinds DNA by 19.1{degrees} {+/-} 0.7{degrees}. We complement the MT data with a spectroscopic characterization of SYBR Gold fluorescence upon addition to DNA. The data are well described by a global binding model for dye concentrations [≤]1 M, with binding parameters that quantitatively agree with the MT results. The fluorescence signal increases linearly with the number of intercalated SYBR Gold molecules. At dye concentrations >1 M, fluorescence quenching and inner filter effects become relevant and it is required to correct the SYBR Gold fluorescence signals for quantitative assessment of DNA concentrations. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold.

biophysics↗

bioRxiv2020

Designed Anchoring Geometries Determine Lifetimes of Biotin-Streptavidin Bonds under Constant Load and Enable Ultra-Stable Coupling

The small molecule biotin and the homotetrameric protein streptavidin (SA) form a stable and robust complex that plays a pivotal role in many biotechnological and medical applications. In particular, the biotin-streptavidin linkage is frequently used in single molecule force spectroscopy (SMFS) experiments. Recent data suggest that biotin-streptavidin bonds show strong directional dependence and a broad range of multi-exponential lifetimes under load. Here, we investigate engineered SA variants with different valencies and a unique tethering point under constant forces using a magnetic tweezer assay. We observed two orders-of-magnitude differences in the lifetimes, which we attribute to the distinct force loading geometries in the different SA variants. We identified an especially long-lived tethering geometry that will facilitate ultra-stable SMFS experiments and pave the way for new biotechnological applications.

biophysics↗

bioRxiv2020

High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes

Nucleosomes, the fundamental units of chromatin, regulate readout and expression of eukaryotic genomes. Single-molecule experiments have revealed force-induced transient nucleosome accessibility, but a high-resolution unwrapping landscape in the absence of external forces is currently lacking. Here, we introduce a high-throughput pipeline for the analysis of nucleosome conformations based on atomic force microscopy and automated, multi-parameter image analysis. Our data set of ~10,000 nucleosomes reveals multiple unwrapping states corresponding to steps of 5 bp DNA. For canonical H3 nucleosomes, we observe that dissociation from one side impedes unwrapping from the other side, but unlike to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes, likely due to their shorter N-terminal -helix. Finally, our results reconcile previously conflicting findings about the differences in height between H3 and CENP-A nucleosomes. We expect our approach to enable critical insights into epigenetic regulation of nucleosome structure and stability.

biophysics↗

bioRxiv2019

Cellular and Molecular Probing of Intact Transparent Human Organs

Optical tissue transparency permits cellular and molecular investigation of complex tissues in 3D, a fundamental need in biomedical sciences. Adult human organs are particularly challenging for this approach, owing to the accumulation of dense and sturdy molecules in decades-aged human tissues. Here, we introduce SHANEL method utilizing a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to generate the first intact transparent adult human brain and kidney, and perform 3D histology using antibodies and dyes in centimeters depth. Thereby, we revealed structural details of sclera, iris and suspensory ligament in the human eye, and the vessels and glomeruli in the human kidney. We also applied SHANEL on transgenic pig organs to map complex structures of EGFP expressing beta cells in >10 cm size pancreas. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of intact large mammalian organs.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC=\"FIGDIR/small/643908v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (89K):\norg.highwire.dtl.DTLVardef@14fea50org.highwire.dtl.DTLVardef@1a04274org.highwire.dtl.DTLVardef@6ae1fforg.highwire.dtl.DTLVardef@11b31b0_HPS_FORMAT_FIGEXP M_FIG C_FIG Supplementary Movies of SHANEL are available at http://discotechnologies.org/SHANEL/

cell biology↗

bioRxiv2018

Modular, ultra-stable, and highly parallel protein force spectroscopy in magnetic tweezers using peptide linkers

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force-regulation, and function. Here, we present a modular magnetic tweezers force spectroscopy approach that uses elastin-like polypeptide linkers to provide a high yield of protein tethers. Our approach extends protein force spectroscopy into the low force (<1 pN) regime and enables ultra-stable measurements on many molecules in parallel. We present (un-)folding data for the single protein domain ddFLN4 and for the large multi-domain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. The measurements reveal exponential force-dependencies of unfolding and refolding rates, directly resolve the stabilization of the VWF A2 domain by Ca2+, and discover transitions in the VWF C-domain stem at low forces that likely constitute the first steps of VWF activation in vivo. Our modular attachment approach will enable precise and multiplexed force spectroscopy measurements for a wide range of proteins in the physiologically relevant force regime.

biophysics↗

bioRxiv2018

The Dynamics and Energy Landscape of DNA Plectoneme Nucleation

DNA buckling is the fundamental step for plectoneme nucleation and supercoil dynamics that are critical in the processing of genetic information. Here we systematically quantify DNA buckling dynamics using high-speed magnetic tweezers. Buckling times are [~]10-100 ms and depend exponentially on both applied force and twist. By deconvolving measured time traces with the instrument response, we reconstruct full 2D extension-twist energy landscapes of the buckling transition that reveal an asymmetry between the pre- and post-buckling states and suggest a highly bend transition state conformation.

biophysics↗