Biology subjects

Schmid, S.

Publications and source records attributed to Schmid, S..

26 records · Page 2Linked to original sources

bioRxiv2022

Can DyeCycling break the photobleaching limit in single-molecule FRET?

Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a prohibitively narrow temporal bandwidth of [≤] 3 orders of magnitude. Here, we introduce DyeCycling, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.

biophysics↗

bioRxiv2021

GPR55 in B cells limits atherosclerosis development and regulates plasma cell maturation

Identifying novel pathways regulating the adaptive immune response in chronic inflammatory diseases such as atherosclerosis is of particular interest in view of developing new therapeutic drugs. Here we report that the lipid receptor GPR55 is highly expressed by splenic B cells and inversely correlates with atheroma plaque size in mice. In human carotid endarterectomy specimen, GPR55 transcript levels were significantly lower in unstable compared to stable carotid plaques. To study the impact of GPR55 deficiency in atherosclerosis, we crossed Gpr55 knockout mice with apolipoprotein E (ApoE) knockout mice and subjected the mice to Western diet for 4 to 16 weeks. Compared to ApoE-/- controls, ApoE-/-Gpr55-/- mice developed larger plaques with increased necrotic core size, associated with elevated circulating and aortic leukocyte counts. Flow cytometry, immunofluorescence and RNA-sequencing analysis of splenic B cells in these mice revealed a hyperactivated B cell phenotype with disturbed plasma cell maturation and immunoglobulin (Ig)G antibody overproduction. The specific contribution of B cell GPR55 in atherosclerosis was further studied in mixed Gpr55-/-/{micro}MT bone marrow chimeras on low density receptor deficiency (Ldlr-/-) background, revealing that B-cell specific depletion of Gpr55 was sufficient to promote plaque development. Conversely, adoptive transfer of wildtype B cells into ApoE-/-Gpr55-/- mice blunted the proatherogenic phenotype. In vitro stimulation of splenocytes with the endogenous GPR55 ligand LPI promoted plasma cell proliferation and enhanced B cell activation marker expression, which was inhibited by the GPR55 antagonist CID16020046. Collectively, these discoveries provide new evidence for GPR55 as key modulator of the adaptive immune response in atherosclerosis. Targeting GPR55 could be useful to limit inflammation and plaque progression in patients suffering from atherosclerosis.

immunology↗

bioRxiv2021

Inferring kinetic rate constants from single-molecule FRET trajectories - a blind benchmark of kinetic analysis tools

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

biophysics↗

bioRxiv2021

Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations

Many strategies have been pursued to trap and monitor single proteins over time in order to detect the molecular mechanisms of these essential nanomachines. Single protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection based on ion currents. Here we present the Nanopore Electro-Osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with sub-millisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtraps ability to sensitively distinguish proteins based on size and shape, and discriminate nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.

biophysics↗

bioRxiv2019

Comparing current noise in biological and solid-state nanopores

Nanopores bear great potential as single-molecule tools for bioanalytical sensing and sequencing, due to their exceptional sensing capabilities, high-throughput, and low cost. The detection principle relies on detecting small differences in the ionic current as biomolecules traverse the nanopore. A major bottleneck for the further progress of this technology is the noise that is present in the ionic current recordings, because it limits the signal-to-noise ratio and thereby the effective time resolution of the experiment. Here, we review the main types of noise at low and high frequencies and discuss the underlying physics. Moreover, we compare biological and solid-state nanopores in terms of the signal-to-noise ratio (SNR), the important figure of merit, by measuring free translocations of a short ssDNA through a selected set of nanopores under typical experimental conditions. We find that SiNx solid-state nanopores provide the highest SNR, due to the large currents at which they can be operated and the relatively low noise at high frequencies. However, the real game-changer for many applications is a controlled slowdown of the translocation speed, which for MspA was shown to increase the SNR >160-fold. Finally, we discuss practical approaches for lowering the noise for optimal experimental performance and further development of the nanopore technology.

biophysics↗

bioRxiv2019

Same Equilibrium. Different Kinetics. Protein Functional Consequences.

In a living cell, protein function is regulated in several ways, including post-translational modifications (PTMs), protein-protein interaction, or by the global environment (e.g. crowding or phase separation). While site-specific PTMs act very locally on the protein, specific protein interactions typically affect larger (sub-)domains, and global changes affect the whole protein in non-specific ways. Herein, we directly observe protein regulation in three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational equilibria, kinetics and protein function. Interestingly using single-molecule FRET, we find that similar functional and conformational steady-states are caused by completely different underlying kinetics. Solving the complete kinetic rate model allows us to disentangle specific and non-specific effects controlling Hsp90s ATPase function, which has remained a puzzle up to this day. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results highlight how cellular protein regulation works by fine-tuning the conformational state space of proteins. SignificanceProteins are perceived more and more as dynamic systems whose function depends critically on local and global flexibility. While 3D structures of proteins are frequently available today, our models often lack the time component, namely rate constants that determine protein function and regulation. Here we used single-molecule FRET to elucidate how the chaperone protein Hsp90 is regulated on various levels, locally and globally. We find that ATPase stimulation occurs not only through specific interactions, but also non-specifically by reducing non-productive conformational flexibility; i.e. by changing kinetics rather than thermodynamics. Our work introduces stimulation through conformational confinement as a general mechanistic concept. We anticipate that this concept plays an important role in protein regulation, phase separation, and in dynamic protein systems in general.

biophysics↗

bioRxiv2018

nsearch: An open source C++ library for processing and similarity searching of next-generation sequencing data

Advancements in DNA sequencing technologies rapidly change the landscape of modern biology. The novel next-generation sequencing (NGS) applications often have special requirements regarding experimental data processing. Software tools developed and used for novel applications are generally designed for specific use cases and as such may be difficult to adapt to new uses. Simultaneously, software tools designed to be general are often difficult to adapt to special use cases.\n\nHere, we present nsearch, a modern open source C++11 library and command-line tool for biological sequence data processing. nsearch offers commonly used components for handling of biological sequences including paired-end read merging, quality filtering and sequence similarity searching. nsearch can either be embedded natively into other C++ applications or be packaged as a standalone executable.\n\nFunctionality and performance of nsearch is shown using benchmark data created using the Rfam 13 database. Benchmarking against common general purpose tools USEARCH and VSEARCH demonstrates that nsearch delivers performance comparable these state-of-the-art tools.\n\nnsearch is available on GitHub under the permissive BSD-3-clause license: https://github.com/stevschmid/nsearch

bioinformatics↗

bioRxiv2018

Effects of inhibitors on Hsp90’s conformational dynamics, cochaperone and client interactions

The molecular chaperone and heat-shock protein Hsp90 has become a central target in anti-cancer therapy. Nevertheless, the effect of Hsp90 inhibition is still not understood at the molecular level, preventing a truly rational drug design. Here we report on the effect of the most prominent drug candidates, namely radicicol, geldanamycin, derivatives of purine and novobiocin, on Hsp90s characteristic conformational dynamics and the binding of three interaction partners. Unexpectedly, the global opening and closing transitions are hardly affected by Hsp90 inhibitors. Instead, the conformational equilibrium, as well as the associated kinetic rate constants remain almost untouched. Moreover, we find no significant changes in the binding of the cochaperones Aha1 and p23 nor of the model substrate {Delta}131{Delta}. This holds true for both, competitive and allosteric inhibitors. Therefore, direct inhibition mechanisms, affecting only one molecular interaction, are unlikely. Based on our results, we speculate that the inhibitory action observed in vivo is caused by a combination of subtle effects, which can be used in the search for novel Hsp90 inhibition mechanisms.

biochemistry↗