Biology subjects

Cordes, T.

Publications and source records attributed to Cordes, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2022

Endogenous 1-deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I (HSAN1), resulting in the synthesis and accumulation of 1-deoxysphingolipids. These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine can promote 1-deoxysphingolipid synthesis, they impact numerous other metabolic pathways important for cancer cells. Here we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxysphingolipid toxicity in cancer cells. Both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxysphingolipid synthesis was induced via SPTLC1C133Wexpression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxysphingolipid synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.

cancer biology↗

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

High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization

Many viruses form highly pleomorphic particles; in influenza, these particles range from spheres of ~ 100 nm in diameter to filaments of several microns in length. Virion structure is of interest, not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline to image many thousands of individual influenza virions, gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. We observed broad phenotypic variability in filament size, and Fourier transform analysis of super resolution images demonstrated no generalized common spatial frequency patterning of HA or NA on the virion surface, suggesting a model of virus particle assembly where the release of progeny filaments from cells occurs in a stochastic way. Finally, we showed that in long filaments, viral RNP complexes are located preferentially within Archetti bodies, suggesting that these structures may play a role in virus transmission. Our approach therefore offers exciting new insights into influenza virus morphology and represents a powerful technique that is easily extendable to the study of pleomorphism in other pathogenic viruses.

microbiology↗

bioRxiv2021

NaCT (SLC13A5) facilitates citrate import and metabolism under nutrient-limited conditions

Citrate lies at a critical node of metabolism linking tricarboxylic acid metabolism and fatty acid synthesis via acetyl-coenzyme A. Recent studies have linked the sodium citrate transporter (NaCT), encoded by SLC13A5, to dysregulated hepatic metabolism and pediatric epilepsy. To examine how NaCT-mediated citrate metabolism contributes to the pathophysiology of these diseases we applied 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cell lines and primary rat cortical neurons. Exogenous citrate contributed to intermediary metabolism at appreciable levels only under hypoxic conditions. In the absence of glutamine, citrate supplementation increased de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromised citrate uptake and catabolism. Citrate supplementation rescued Huh7 cell viability in response to glutamine deprivation and Zn2+ treatment, and these effects were mitigated by NaCT deficiency. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient limited conditions and may facilitate resistance to metal toxicity.

cell biology↗

bioRxiv2021

Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas

Deconstructing tissue-specific effects of genes and variants on proliferative advantage is critical to understanding cellular transformation and to systematic selection of cancer therapeutics. Dissecting these specificities at scale requires integrated methods for multiplexed genetic screens tracking fitness across time, across human cell types, and in a suitable cellular niche since functional differences also depend on physiological cues. Towards this, we present a novel approach, harnessing single-cell cancer driver screens in teratomas coupled with hit enrichment by serial teratoma reinjection, to simultaneously screen drivers across multiple lineages in vivo. Using this system, we analyzed population shifts and lineage-specific enrichment for 51 cancer associated genes and gene variants, profiling over 100,000 cells spanning over 20 lineages, across two rounds of serially injected teratomas. We confirmed that c-MYC alone or combined with myristoylated AKT1 potently drives proliferation in progenitor neural lineages, demonstrating signatures of malignancy. These drivers directed teratoma development to lineages representative of pediatric tumors such as medulloblastoma and rhabdomyosarcoma. Additionally, mutant MEK1S218D/S222D provides a proliferative advantage in mesenchymal lineages like fibroblasts. Our method provides a powerful new platform for multi-lineage longitudinal study of oncogenesis.

systems biology↗

bioRxiv2020

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid, and asparagine in Gram-positive bacteria. It features two extracytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from L. lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Forster-resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously. HIGHLIGHTSO_LIResolved crystal structure of tandem SBD1-2 of GlnPQ from Lactococcus lactis C_LIO_LIConformational states and ligand binding affinities of individual domains SBD1 and SBD2 are similar to tandem SBD1-2 C_LIO_LINo cooperative effects are seen for different ligands for SBDs in the tandem C_LIO_LIProof of concept experiments show that PIFE-FRET can monitor SBD conformations and protein-protein interaction simultaneously C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/423572v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1fa36f7org.highwire.dtl.DTLVardef@2e973forg.highwire.dtl.DTLVardef@cbae51org.highwire.dtl.DTLVardef@2185cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Triggering closure of a sialic acid TRAP transporter substrate binding protein through binding of natural or artificial substrates

The pathogens Vibrio cholerae and Haemophilus influenzae use tripartite ATP-independent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues. They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open- to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter from V. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.

biophysics↗

bioRxiv2020

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) and single molecule Forster resonance energy transfer spectroscopy (smFRET) are recent additions to the toolbox of integrative structural biology. Both methods are frequently used to visualize conformational changes and to determine nanometer-scale distances in biomacromolecules including proteins and nucleic acids. A prerequisite for the application of PELDOR/DEER and smFRET is the presence of suitable spin centers or fluorophores in the target molecule, which are usually introduced via chemical biology methods. The application portfolio of the two methods is overlapping: each allows determination of distances, to monitor distance changes and to visualize conformational heterogeneity and -dynamics. Both methods can provide qualitative information that facilitates mechanistic understanding, for instance on conformational changes, as well as quantitative data for structural modelling. Despite their broad application, a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET is still missing and we set out here to fill this gap. For this purpose, we prepared a library of double cysteine mutants of three well-studied substrate binding proteins that undergo large-scale conformational changes upon ligand binding. The distances between the introduced spin- or fluorescence labels were determined via PELDOR/DEER and smFRET, using established standard experimental protocols and data analysis routines. The experiments were conducted in the presence and absence of the natural ligands to investigate how well the ligand-induced conformational changes could be detected by the two methods. Overall, we found good agreement for the determined distances, yet some surprising inconsistencies occurred. In our set of experiments, we identified the source of discrepancies as the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. Our study highlights strength and weaknesses of both methods and paves the way for a higher confidence in quantitative comparison of PELDOR/DEER and smFRET results in the future.

biophysics↗

bioRxiv2020

Functional self-nonamerization of the Type III translocase chaperone/exported protein receptor

Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante- chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E.coli (EPEC). In both EPEC and E.coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of [~]22 nm with a N- terminal domain connected by a short linker to a C-domain ring structure with a [~]5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 [A] and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with pore surface and ring entry mouth local conformational changes that are also modulated by the ATPase inner stalk. We propose a model that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.

molecular biology↗

bioRxiv2020

Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series

The use of fluorescence techniques has had an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the availability of numerous commercial dyes, but often information about the chemical structures of dyes (and their linkers) are a well-kept secret. This can lead to problems for applications where a knowledge of the dye structure is necessary to predict (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of spectroscopy, mass spectrometry and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we present two possible structures for Alexa Fluor 555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647. A comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes, supports the suggestion that the Alexa Fluor and AF dyes have a high degree of structural similarity. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation demonstrating that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes for smFRET studies, and suggesting their usefulness for other fluorescence applications.

biophysics↗

bioRxiv2020

Evolution of structural dynamics in bilobed proteins

Novel biophysical tools allow the structural dynamics of proteins, and the regulation of such dynamics by binding partners, to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remains poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the type-II periplasmic binding protein domain family, have undergone divergent evolution leading to modification of their structural dynamics and function. We performed a structural analysis of ~600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Forster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enables distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extra-cytoplasmic transport-related proteins. Structural embellishments of the core created new interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately, altered substrate specificity. Our findings reveal an as yet unrecognized mechanism for the emergence of functional promiscuity during long periods of protein evolution and are applicable to a large number of domain architectures.

biophysics↗

bioRxiv2020

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Forster-resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the type-I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using the methodology, we studied ligand-binding mechanisms as well as ATP and glycine betaine dependences of conformational changes. Our study expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

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

Characterization of fluorescent proteins with intramolecular photostabilization

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Since there are no other natural fluorescent tags with comparable features, the impact of fluorescent proteins for biological research cannot be overemphasized. Despite their importance, their photophysical properties, i.e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties. The approach uses direct conjugation of photostablizing compounds such as triplet-state quenchers or redox-active substances to an organic fluorophore, thereby creating high local concentrations of photostabilizer. Here, we introduce an experimental strategy to screen for the effects of covalently-linked photostabilizers on fluorescent proteins. We recombinantly produced a double cysteine mutant (A206C/L221C) of -GFP for attachment of photostabilizer-maleimides on the {beta}-barrel in close proximity to the chromophore. Whereas labelling with photostabilizers such as Trolox, Nitrophenyl, and Cyclooctatetraene, which are often used for organic fluorophores, had no effect on -GFP-photostability, a substantial increase of photostability was found upon conjugation of -GFP to an azobenzene derivative. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the near-UV and blue spectral range. Our study paves the way towards the development and design of a second generation of fluorescent proteins with photostabilizers placed directly in the protein barrel by methods such as unnatural amino acid incorporation.

biophysics↗

bioRxiv2019

Single-molecule observation of ligand binding and conformational changes in FeuA

The specific binding of ligands by proteins and the coupling of this process to conformational changes are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ABC importer FeuBC, which is involved in iron uptake in bacteria. The conformational dynamics of FeuA was assessed via Forster resonance energy transfer (FRET), whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation without the involvement of the ligand. This shows that ligand interactions are not required for conformational changes in FeuA. However, ligand interactions accelerate the conformational change 10000-fold and temporally stabilize the formed conformation 250-fold.\n\nSIGNIFICANCE STATEMENTLigand binding and the coupling of this process to conformational changes in proteins are fundamental to their function. We developed a single-molecule approach that allows the simultaneous observation of ligand binding and conformational changes in the substrate-binding protein FeuA. This allows to directly observe the ligand binding process, ligand-driven conformational changes as well as rare short-lived conformational transitions that are uncoupled from the ligand. These findings provide insight into the fundamental relation between ligand-protein interactions and conformational changes. Our findings are, however, not only of interest to understand protein function, but the developed data analysis procedure allows the determination of (relative) distance changes in single-molecule FRET experiments, for situations in which donor and acceptor fluorophore are influenced by quenching processes.

biophysics↗

bioRxiv2019

ColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data

Single-molecule fluorescence microscopy studies of bacteria provide unique insights into the mechanisms of cellular processes and protein machineries in ways that are unrivalled by any other technique. With the cost of microscopes dropping and the availability of fully automated microscopes, the volume of microscopy data produced has increased tremendously. These developments have moved the bottleneck of throughput from image acquisition and sample preparation to data analysis. Furthermore, requirements for analysis procedures have become more stringent given the requirement of various journals to make data and analysis procedures available. To address this we have developed a new data analysis package for analysis of fluorescence microscopy data of rod-like cells. Our software ColiCoords structures microscopy data at the single-cell level and implements a coordinate system describing each cell. This allows for the transformation of Cartesian coordinates of both cellular images (e.g. from transmission light or fluorescence microscopy) and single-molecule localization microscopy (SMLM) data to cellular coordinates. Using this transformation, many cells can be combined to increase the statistical significance of fluorescence microscopy datasets of any kind. Coli-Coords is open source, implemented in the programming language Python, and is extensively documented. This allows for modifications for specific needs or to inspect and publish data analysis procedures. By providing a format that allows for easy sharing of code and associated data, we intend to promote open and reproducible research.\n\nThe source code and documentation can be found via the projects GitHub page.

bioinformatics↗

bioRxiv2019

Conformational and dynamical plasticity in substrate-binding proteins underlies selective transport in ABC importers

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open-to a closed-conformation upon substrate binding providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.

biophysics↗