Publications by authors named "Jelle Hendrix"

49 Publications

A topological switch in CFTR modulates channel activity and sensitivity to unfolding.

Nat Chem Biol 2021 09 2;17(9):989-997. Epub 2021 Aug 2.

SFMB, Université Libre de Bruxelles, Brussels, Belgium.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.
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http://dx.doi.org/10.1038/s41589-021-00844-0DOI Listing
September 2021

The polymeric glyco-linker controls the signal outputs for plasmonic gold nanorod biosensors due to biocorona formation.

Nanoscale 2021 Jun;13(24):10837-10848

Health Unit, Flemish Institute for Technological Research (VITO), Boeretang 200, Mol, BE-2400, Belgium.

Gold nanorods (GNRs) are a promising platform for nanoplasmonic biosensing. The localised surface plasmon resonance (LSPR) peak of GNRs is located in the near-infrared optical window and is sensitive to local binding events, enabling label-free detection of biomarkers in complex biological fluids. A key challenge in the development of such sensors is achieving target affinity and selectivity, while both minimizing non-specific binding and maintaining colloidal stability. Herein, we reveal how GNRs decorated with galactosamine-terminated polymer ligands display significantly different binding responses in buffer compared to serum, due to biocorona formation, and how biocorona displacement due to lectin binding plays a key role in their optical responses. GNRs were coated with either poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) or poly(N-hydroxyethyl acrylamide) (PHEA) prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerisation and end-functionalised with galactosamine (Gal) as the lectin-targeting unit. In buffer Gal-PHEA-coated GNRs aggregated upon soybean agglutinin (SBA) addition, whereas Gal-PHPMA-coated GNRs exhibited a red-shift of the LSPR spectrum without aggregation. In contrast, when incubated in serum Gal-PHPMA-coated nanorods showed no binding response, while Gal-PHEA GNRs exhibited a dose-dependent blue-shift of the LSPR peak, which is the opposite direction (red-shift) to what was observed in buffer. This differential behaviour was attributed to biocorona formation onto both polymer-coated GNRs, shown by differential centrifugal sedimentation and nanoparticle tracking analysis. Upon addition of SBA to the Gal-PHEA coated nanorods, signal was generated due to displacement of weakly-bound biocorona components by lectin binding. However, in the case of Gal-PHPMA which had a thicker corona, attributed to lower polymer grafting densities, addition of SBA did not lead to biocorona displacement and there was no signal output. These results show that plasmonic optical responses in complex biological media can be significantly affected by biocorona formation, and that biocorona formation itself does not prevent sensing so long as its exact nature (e.g. 'hard versus soft') is tuned.
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http://dx.doi.org/10.1039/d1nr01548fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223873PMC
June 2021

Quantification of FRET-induced angular displacement by monitoring sensitized acceptor anisotropy using a dim fluorescent donor.

Nat Commun 2021 05 5;12(1):2541. Epub 2021 May 5.

Laboratory for Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Heverlee, Belgium.

Förster resonance energy transfer (FRET) between fluorescent proteins has become a common platform for designing genetically encoded biosensors. For live cell imaging, the acceptor-to-donor intensity ratio is most commonly used to readout FRET efficiency, which largely depends on the proximity between donor and acceptor. Here, we introduce an anisotropy-based mode of FRET detection (FADED: FRET-induced Angular Displacement Evaluation via Dim donor), which probes for relative orientation rather than proximity alteration. A key element in this technique is suppression of donor bleed-through, which allows measuring purer sensitized acceptor anisotropy. This is achieved by developing Geuda Sapphire, a low-quantum-yield FRET-competent fluorescent protein donor. As a proof of principle, Ca sensors were designed using calmodulin as a sensing domain, showing sigmoidal dose response to Ca. By monitoring the anisotropy, a Ca rise in living HeLa cells is observed upon histamine challenging. We conclude that FADED provides a method for quantifying the angular displacement via FRET.
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http://dx.doi.org/10.1038/s41467-021-22816-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099864PMC
May 2021

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Elife 2021 03 29;10. Epub 2021 Mar 29.

Departments of Biology and Chemistry, Johannes Gutenberg University, Mainz, Germany.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.
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http://dx.doi.org/10.7554/eLife.60416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007216PMC
March 2021

Unsupervised Machine Learning-Based Clustering of Nanosized Fluorescent Extracellular Vesicles.

Small 2021 02 15;17(5):e2006786. Epub 2021 Jan 15.

Biomedical Research Institute (BIOMED), Hasselt University, Martelarenlaan 42, Hasselt, 3500, Belgium.

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.
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http://dx.doi.org/10.1002/smll.202006786DOI Listing
February 2021

Imaging the Replication of Single Viruses: Lessons Learned from HIV and Future Challenges To Overcome.

ACS Nano 2020 09 21;14(9):10775-10783. Epub 2020 Aug 21.

Molecular Imaging and Photonics, Chemistry Department, KU Leuven, B-3001 Heverlee, Flanders, Belgium.

The molecular composition of viral particles indicates that a single virion is capable of initiating an infection. However, the majority of viruses that come into contact with cells fails to infect them. Understanding what makes one viral particle more successful than others requires visualizing the infection process directly in living cells, one virion at a time. In this Perspective, we explain how single-virus imaging using fluorescence microscopy can provide answers to unsolved questions in virology. We discuss fluorescent labeling of virus particles, resolution at the subviral and molecular levels, tracking in living cells, and imaging of interactions between viral and host proteins. We end this Perspective with a set of remaining questions in understanding the life cycle of retroviruses and how imaging a single virus can help researchers address these questions. Although we use examples from the HIV field, these methods are of value for the study of other viruses as well.
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http://dx.doi.org/10.1021/acsnano.0c06369DOI Listing
September 2020

Correction to: LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions.

Retrovirology 2020 07 29;17(1):22. Epub 2020 Jul 29.

Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Molecular Virology and Gene Therapy, KU Leuven, Kapucijnenvoer 33, 3000, Louvain, Flanders, Belgium.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s12977-020-00530-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389342PMC
July 2020

Live Cell Imaging Demonstrates Multiple Routes Toward a STAT1 Gain-of-Function Phenotype.

Front Immunol 2020 9;11:1114. Epub 2020 Jun 9.

Department of Microbiology, Immunology and Transplantation, Allergy and Clinical Immunology Research Group, KU Leuven, Leuven, Belgium.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that are spread over the protein and studied their dynamic behavior in U3A and HeLa cell lines. All GOF mutants showed increased STAT1 phosphorylation compared to STAT1 WT. Real-time imaging demonstrated three underlying mechanisms for STAT1 GOF: (i) R274W showed a faster nuclear accumulation, (ii) both R321S and N574I showed a reduced nuclear mobility and slower dephosphorylation, whereas (iii) T419R was near-immobile in the nucleus, potentially due to enhanced binding to chromatin.
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http://dx.doi.org/10.3389/fimmu.2020.01114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296103PMC
April 2021

A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes.

Nat Chem Biol 2020 08 11;16(8):834-840. Epub 2020 May 11.

Cellular and Molecular Microbiology, Faculté des Sciences, Université Libre de Bruxelles, Brussels, Belgium.

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (Rel). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of Rel (Rel) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.
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http://dx.doi.org/10.1038/s41589-020-0520-2DOI Listing
August 2020

Fluorescence microscopy data for quantitative mobility and interaction analysis of proteins in living cells.

Data Brief 2020 Apr 28;29:105348. Epub 2020 Feb 28.

Hasselt University, Advanced Optical Microscopy Centre and Biomedical Research Institute, Dynamic Bioimaging Lab, Diepenbeek, Belgium.

The data provided with this paper are image series of slowly diffusing GlyRa3 molecules, linked to either eGFP or mCherry fluorescent proteins, at the membrane of HEK cells, acquired on a Zeiss LSM880 confocal laser scanning microscope. Raster spectral image cross-correlation spectroscopy (RSICS) is applied to the data, a technique that exploits intensity fluctuations in confocal image series recorded using a spectral detector to study the diffusion and concentration of molecules, and interactions between them. First, spectral filters are created from reference image series containing GlyRa3 labeled with a single fluorophore. Once experimental data containing GlyRa3 labeled with both fluorophores is acquired, single images are either autocorrelated, or the cross-correlation is calculated between two images, each one containing the data for eGFP or mCherry labeled GyRa 3. Data is then fit with a one-component model assuming a two-dimensional Gaussian point spread function to obtain the diffusion coefficient, , and average number of molecules in the focus, . The software package PAM is used to analyze all the acquired data. The data can be used as a reference for artifact-free two-color ccRICS that contains slowly diffusing interacting molecules. Additionally, the analysis workflow described in this paper helps researchers avoid common errors during a RICS experiment.
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http://dx.doi.org/10.1016/j.dib.2020.105348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066057PMC
April 2020

Capsid-Labelled HIV To Investigate the Role of Capsid during Nuclear Import and Integration.

J Virol 2020 03 17;94(7). Epub 2020 Mar 17.

Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium

The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating enhanced green fluorescent protein (eGFP)-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild-type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP-containing complexes. Low numbers of CA-eGFP molecules were located inside the viral core and imported into the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retained both labelled proteins after nuclear entry, implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP-containing complexes were detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP-containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration. HIV-1 capsid protein (CA) builds a conical shell protecting viral genomic RNA inside the virus particles. Upon entry into host cells, this shell disassembles in a process of uncoating, which is coordinated with reverse transcription of viral RNA into DNA. After uncoating, a portion of CA remains associated with the viral DNA and mediates its nuclear import and, potentially, integration into host DNA. In this study, we tagged CA with eGFP to follow its trafficking in host cells and address potential CA roles in the nucleus. We found that while functional viruses import the tagged CA into the nucleus, this capsid protein is not part of integration-competent complexes. The roles of nuclear CA thus remain to be established.
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http://dx.doi.org/10.1128/JVI.01024-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081887PMC
March 2020

Raster Image Correlation Spectroscopy Performance Evaluation.

Biophys J 2019 11 10;117(10):1900-1914. Epub 2019 Oct 10.

Department of Mathematical Sciences, Chalmers University of Technology and University of Gothenburg, Gothenburg, Sweden.

Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracting the mobility, concentration, and stoichiometry of diffusing fluorescent molecules from confocal image stacks. The method works by calculating a spatial correlation function for each image and analyzing the average of those by model fitting. Rules of thumb exist for RICS image acquisitioning, yet a rigorous theoretical approach to predict the accuracy and precision of the recovered parameters has been lacking. We outline explicit expressions to reveal the dependence of RICS results on experimental parameters. In terms of imaging settings, we observed that a twofold decrease of the pixel size, e.g., from 100 to 50 nm, decreases the error on the translational diffusion constant (D) between three- and fivefold. For D = 1 μm s, a typical value for intracellular measurements, ∼25-fold lower mean-squared relative error was obtained when the optimal scan speed was used, although more drastic improvements were observed for other values of D. We proposed a slightly modified RICS calculation that allows correcting for the significant bias of the autocorrelation function at small (≪50 × 50 pixels) sizes of the region of interest. In terms of sample properties, at molecular brightness E = 100 kHz and higher, RICS data quality was sufficient using as little as 20 images, whereas the optimal number of frames for lower E scaled pro rata. RICS data quality was constant over the nM-μM concentration range. We developed a bootstrap-based confidence interval of D that outperformed the classical least-squares approach in terms of coverage probability of the true value of D. We validated the theory via in vitro experiments of enhanced green fluorescent protein at different buffer viscosities. Finally, we outline robust practical guidelines and provide free software to simulate the parameter effects on recovery of the diffusion coefficient.
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http://dx.doi.org/10.1016/j.bpj.2019.09.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018992PMC
November 2019

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics.

Structure 2019 01 21;27(1):90-101.e6. Epub 2018 Nov 21.

KU Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory for Molecular Bacteriology, Herestraat 49, Gasthuisberg Campus, B-3000 Leuven, Belgium. Electronic address:

SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Förster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis.
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http://dx.doi.org/10.1016/j.str.2018.10.006DOI Listing
January 2019

Post-mitotic BET-induced reshaping of integrase quaternary structure supports wild-type MLV integration.

Nucleic Acids Res 2019 02;47(3):1195-1210

Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium.

The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.
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http://dx.doi.org/10.1093/nar/gky1157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379647PMC
February 2019

Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome of .

Proc Natl Acad Sci U S A 2018 11 14;115(48):E11274-E11283. Epub 2018 Nov 14.

Physical Chemistry, Department of Chemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany;

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin-cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as "beads on a string," we propose that cohesin-cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.
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http://dx.doi.org/10.1073/pnas.1809283115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275499PMC
November 2018

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nat Methods 2018 09 31;15(9):669-676. Epub 2018 Aug 31.

Institute for Biophysics, Ulm University, Ulm, Germany.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.
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http://dx.doi.org/10.1038/s41592-018-0085-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121742PMC
September 2018

Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication.

Viruses 2018 05 10;10(5). Epub 2018 May 10.

Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics Division, Chemistry Department, KU Leuven, B-3001 Leuven, Belgium.

Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that we employ routinely to investigate viruses. We provide a brief overview of the microscopy hardware needed and discuss the different methods and their application. In particular, we review how we applied (i) single-molecule Förster resonance energy transfer (smFRET) to probe the subviral human immunodeficiency virus (HIV-1) integrase (IN) quaternary structure; (ii) single particle tracking to study interactions of the simian virus 40 with membranes; (iii) 3D confocal microscopy and smFRET to quantify the HIV-1 pre-integration complex content and quaternary structure; (iv) image correlation spectroscopy to quantify the cytosolic HIV-1 Gag assembly, and finally; (v) super-resolution microscopy to characterize the interaction of HIV-1 with tetherin during assembly. We hope this review is an incentive for setting up and applying similar single-virus imaging studies in daily virology practice.
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http://dx.doi.org/10.3390/v10050250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5977243PMC
May 2018

PAM: A Framework for Integrated Analysis of Imaging, Single-Molecule, and Ensemble Fluorescence Data.

Biophys J 2018 04;114(7):1518-1528

Department of Physical Chemistry, Center for Integrated Protein Science Munich (CIPSM), Nanosystems Initiative Munich (NIM) and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität, Munich, Germany. Electronic address:

Fluorescence microscopy and spectroscopy data hold a wealth of information on the investigated molecules, structures, or organisms. Nowadays, the same fluorescence data set can be analyzed in many ways to extract different properties of the measured sample. Yet, doing so remains slow and cumbersome, often requiring incompatible software packages. Here, we present PAM (pulsed interleaved excitation analysis with MATLAB), an open-source software package written in MATLAB that offers a simple and efficient workflow through its graphical user interface. PAM is a framework for integrated and robust analysis of fluorescence ensemble, single-molecule, and imaging data. Although it was originally developed for the analysis of pulsed interleaved excitation experiments, PAM has since been extended to support most types of data collection modalities. It combines a multitude of powerful analysis algorithms, ranging from time- and space-correlation analysis, over single-molecule burst analysis, to lifetime imaging microscopy, while offering intrinsic support for multicolor experiments. We illustrate the key concepts and workflow of the software by discussing data handling and sorting and provide step-by-step descriptions for the individual usage cases.
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http://dx.doi.org/10.1016/j.bpj.2018.02.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954487PMC
April 2018

Phosphorylation decelerates conformational dynamics in bacterial translation elongation factors.

Sci Adv 2018 03 14;4(3):eaap9714. Epub 2018 Mar 14.

Cellular and Molecular Microbiology, Department of Molecular Biology, Université Libre de Bruxelles, Brussels, Belgium.

Bacterial protein synthesis is intricately connected to metabolic rate. One of the ways in which bacteria respond to environmental stress is through posttranslational modifications of translation factors. Translation elongation factor Tu (EF-Tu) is methylated and phosphorylated in response to nutrient starvation upon entering stationary phase, and its phosphorylation is a crucial step in the pathway toward sporulation. We analyze how phosphorylation leads to inactivation of EF-Tu. We provide structural and biophysical evidence that phosphorylation of EF-Tu at T382 acts as an efficient switch that turns off protein synthesis by decoupling nucleotide binding from the EF-Tu conformational cycle. Direct modifications of the EF-Tu switch I region or modifications in other regions stabilizing the β-hairpin state of switch I result in an effective allosteric trap that restricts the normal dynamics of EF-Tu and enables the evasion of the control exerted by nucleotides on G proteins. These results highlight stabilization of a phosphorylation-induced conformational trap as an essential mechanism for phosphoregulation of bacterial translation and metabolism. We propose that this mechanism may lead to the multisite phosphorylation state observed during dormancy and stationary phase.
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http://dx.doi.org/10.1126/sciadv.aap9714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851678PMC
March 2018

Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule Förster Resonance Energy Transfer Experiments.

J Phys Chem B 2018 04 5;122(15):4249-4266. Epub 2018 Apr 5.

Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry , KU Leuven , Celestijnenlaan 200F , B-3001 Leuven , Belgium.

Förster resonance energy transfer (FRET) is a powerful tool to probe molecular interactions, activity, analytes, forces, and structure. Single-molecule (sm)FRET additionally allows real-time quantifications of conformation and conformational dynamics. smFRET robustness critically depends on the employed dyes, yet a systematic comparison of different dye pairs is lacking. Here, we evaluated blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647, StarRed, and Atto655) dyes using confocal smFRET spectroscopy on freely diffusing double-stranded (ds)DNA molecules. Via ensemble analyses (correlation, lifetime, and anisotropy) of single-labeled dsDNA, we find that Alexa488 and Atto647N are overall the better dyes, although the latter interacts with DNA. Via burstwise analyses of double-labeled dsDNA with interdye distances spanning the complete FRET-sensitive range (3.5-9 nm), we show that none of the dye pairs stands out: distance accuracies were generally <1 nm and precision was ∼0.5 nm. Finally, excitation of photoblinking dyes such as Alexa647 influences their fluorescence quantum yield, which has to be taken into account in distance measurements and leads to FRET dynamics. Although dye performance might differ in experiments on immobilized molecules, our combined ensemble and single-molecule approach is a robust characterization tool for all types of smFRET experiments. This is especially important when smFRET is used for atomic-scale distance measurements.
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http://dx.doi.org/10.1021/acs.jpcb.8b00108DOI Listing
April 2018

Crosstalk-free multicolor RICS using spectral weighting.

Methods 2018 05 22;140-141:97-111. Epub 2018 Feb 22.

Dynamic Bioimaging Lab, Hasselt University, Diepenbeek, Belgium; Advanced Optical Microscopy Centre, Biomedical research institute (BIOMED), Hasselt University, Diepenbeek, Belgium; Molecular Imaging and Photonics, Chemistry Department, KU Leuven, Heverlee, Belgium. Electronic address:

Raster image cross-correlation spectroscopy (ccRICS) can be used to quantify the interaction affinities between diffusing molecules by analyzing the fluctuations between two-color confocal images. Spectral crosstalk compromises the quantitative analysis of ccRICS experiments, limiting multicolor implementations to dyes with well-separated emission spectra. Here, we remove this restriction by introducing raster spectral image correlation spectroscopy (RSICS), which employs statistical filtering based on spectral information to quantitatively separate signals of fluorophores during spatial correlation analysis. We investigate the performance of RSICS by testing how different levels of spectral overlap or different relative signal intensities affect the correlation function and analyze the influence of statistical filter quality. We apply RSICS in vitro to resolve dyes with very similar emission spectra, and carry out RSICS in live cells to simultaneously analyze the diffusion of molecules carrying three different fluorescent protein labels (eGFP, Venus and mCherry). Finally, we successfully apply statistical weighting to data that was recorded with only a single detection channel per fluorophore, highlighting the general applicability of this method to data acquired with any type of multicolor detection. In conclusion, RSICS enables artifact-free quantitative analysis of concentrations, mobility and interactions of multiple species labeled with different fluorophores. It can be performed on commercial laser scanning microscopes, and the algorithm can be easily extended to other image correlation methods. Thus, RSICS opens the door to quantitative multicolor fluctuation analyses of complex (bio-) molecular systems.
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http://dx.doi.org/10.1016/j.ymeth.2018.01.022DOI Listing
May 2018

Bap (Sil1) regulates the molecular chaperone BiP by coupling release of nucleotide and substrate.

Nat Struct Mol Biol 2018 01 1;25(1):90-100. Epub 2018 Jan 1.

Center for Integrated Protein Science Munich at the Department of Chemistry, Technical University of Munich, Garching, Germany.

BiP is the endoplasmic member of the Hsp70 family. BiP is regulated by several co-chaperones including the nucleotide-exchange factor (NEF) Bap (Sil1 in yeast). Bap is a two-domain protein. The interaction of the Bap C-terminal domain with the BiP ATPase domain is sufficient for its weak NEF activity. However, stimulation of the BiP ATPase activity requires full-length Bap, suggesting a complex interplay of these two factors. Here, single-molecule FRET experiments with mammalian proteins reveal that Bap affects the conformation of both BiP domains, including the lid subdomain, which is important for substrate binding. The largely unstructured Bap N-terminal domain promotes the substrate release from BiP. Thus, Bap is a conformational regulator affecting both nucleotide and substrate interactions. The preferential interaction with BiP in its ADP state places Bap at a late stage of the chaperone cycle, in which it coordinates release of substrate and ADP, thereby resetting BiP for ATP and substrate binding.
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http://dx.doi.org/10.1038/s41594-017-0012-6DOI Listing
January 2018

Hidden States within Disordered Regions of the CcdA Antitoxin Protein.

J Am Chem Soc 2017 02 8;139(7):2693-2701. Epub 2017 Feb 8.

Structural Biology Brussels, Department of Biotechnology, Vrije Universiteit Brussel , B-1050 Brussels, Belgium.

The bacterial toxin-antitoxin system CcdB-CcdA provides a mechanism for the control of cell death and quiescence. The antitoxin protein CcdA is a homodimer composed of two monomers that each contain a folded N-terminal region and an intrinsically disordered C-terminal arm. Binding of the intrinsically disordered C-terminal arm of CcdA to the toxin CcdB prevents CcdB from inhibiting DNA gyrase and thereby averts cell death. Accurate models of the unfolded state of the partially disordered CcdA antitoxin can therefore provide insight into general mechanisms whereby protein disorder regulates events that are crucial to cell survival. Previous structural studies were able to model only two of three distinct structural states, a closed state and an open state, that are adopted by the C-terminal arm of CcdA. Using a combination of free energy simulations, single-pair Förster resonance energy transfer experiments, and existing NMR data, we developed structural models for all three states of the protein. Contrary to prior studies, we find that CcdA samples a previously unknown state where only one of the disordered C-terminal arms makes extensive contacts with the folded N-terminal domain. Moreover, our data suggest that previously unobserved conformational states play a role in regulating antitoxin concentrations and the activity of CcdA's cognate toxin. These data demonstrate that intrinsic disorder in CcdA provides a mechanism for regulating cell fate.
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http://dx.doi.org/10.1021/jacs.6b11450DOI Listing
February 2017

Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry.

Sci Rep 2016 11 10;6:36485. Epub 2016 Nov 10.

Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium.

Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.
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http://dx.doi.org/10.1038/srep36485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103197PMC
November 2016

Arbitrary-Region Raster Image Correlation Spectroscopy.

Biophys J 2016 Oct;111(8):1785-1796

Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.

Combining imaging with correlation spectroscopy, as in raster image correlation spectroscopy (RICS), makes it possible to extract molecular translational diffusion constants and absolute concentrations, and determine intermolecular interactions from single-channel or multicolor confocal laser-scanning microscopy (CLSM) images. Region-specific RICS analysis remains very challenging because correlations are always calculated in a square region-of-interest (ROI). In this study, we describe a generalized image correlation spectroscopy algorithm that accepts arbitrarily shaped ROIs. We show that an image series can be cleaned up before arbitrary-region RICS (ARICS) analysis. We demonstrate the power of ARICS by simultaneously measuring molecular mobility in the cell membrane and the cytosol. Mobility near dynamic subcellular structures can be investigated with ARICS by generating a dynamic ROI. Finally, we derive diffusion and concentration pseudo-maps using the ARICS method. ARICS is a powerful expansion of image correlation spectroscopy with the potential of becoming the new standard for extracting biophysical parameters from confocal fluorescence images.
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http://dx.doi.org/10.1016/j.bpj.2016.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073057PMC
October 2016

Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers.

J Cell Biol 2015 Aug;210(4):629-46

Physical Chemistry, Department of Chemistry, Ludwig Maximilian University of Munich, D-81377 Munich, Germany NanoSystems Initiative Munich (NIM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Munich Center for Integrated Protein Science (CiPSM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Center for Nanoscience (CeNS), Ludwig Maximilian University of Munich, D-81377 Munich, Germany

Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.
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http://dx.doi.org/10.1083/jcb.201504006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539982PMC
August 2015

Hsp90 regulates the dynamics of its cochaperone Sti1 and the transfer of Hsp70 between modules.

Nat Commun 2015 Apr 8;6:6655. Epub 2015 Apr 8.

Center for integrated protein science (CIPSM) at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.

The cochaperone Sti1/Hop physically links Hsp70 and Hsp90. The protein exhibits one binding site for Hsp90 (TPR2A) and two binding sites for Hsp70 (TPR1 and TPR2B). How these sites are used remained enigmatic. Here we show that Sti1 is a dynamic, elongated protein that consists of a flexible N-terminal module, a long linker and a rigid C-terminal module. Binding of Hsp90 and Hsp70 regulates the Sti1 conformation with Hsp90 binding determining with which site Hsp70 interacts. Without Hsp90, Sti1 is more compact and TPR2B is the high-affinity interaction site for Hsp70. In the presence of Hsp90, Hsp70 shifts its preference. The linker connecting the two modules is crucial for the interaction with Hsp70 and for client activation in vivo. Our results suggest that the interaction of Hsp70 with Sti1 is tightly regulated by Hsp90 to assure transfer of Hsp70 between the modules, as a prerequisite for the efficient client handover.
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http://dx.doi.org/10.1038/ncomms7655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403447PMC
April 2015

The lateral membrane organization and dynamics of myelin proteins PLP and MBP are dictated by distinct galactolipids and the extracellular matrix.

PLoS One 2014 8;9(7):e101834. Epub 2014 Jul 8.

Department of Cell Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP's diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101834PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4086962PMC
October 2015
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