DNA-binding proteins utilisedifferent recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarse-grained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy ( = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.
K.J.A.Martens, A.N.Bader, S.Baas, B.Rieger, J.Hohlbein, The Journal of Chemical Physics, 148, 123311, 2018, [link]
We present a fast and model-free 2D and 3Dsingle-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier coefficients in both x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.
For the latest software implementation into thunderSTORM, please follow the [link].
C. Fijen, M. Fontana, S.G. Lemay, K. Mathwig, J.Hohlbein, bioRxiv, 2017, [link]
Single-molecule detection schemesoffer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels. A second design featuring a T-shaped nanochannel enables precise mixing of two different species as well as the continuous observation of chemical reactions. We utilized the design to visualize enzymatically driven DNA synthesis in real time and at the single-molecule level. Based on our results, we are convinced that the versatility and performance of the nanofluidic devices will enable numerous applications in the life sciences.
Hurrah, we finalised the design of our miCube (V0.1)! Details can be found [here] and include part numbers, CAD-drawings and STL files for CNC machining/milling or 3D printing published under a Creative Common license. Big shout out to Sander Baas and Koen Martens. If you have any questions, remarks or ideas, please feel to contact me.
J. Hohlbein and A.N. Kapanidis, Methods in Enzymology: Single-molecule Enzymology Part A & B, published online, 2016, [link] Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and the […]
S.J. Hutten, D.S. Hamers, M.A. an den Toorn, W. van Esse, A. Nolles, C.A. Bücherl, S.C. de Vries, J. Hohlbein, J.W. Borst, PLoS ONE 12(1): e0169905, 2017, [link]
Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation—fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction.
E. Ploetz, E. Lerner, F. Husada, M. Roelfs, S. Chung, J. Hohlbein, S. Weiss, T. Cordes, Scientific Reports,6, 33257 , 2016, [link]
Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of dsDNA following its interaction with unlabelled proteins (BamHI, EcoRV, T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.
Update: Follow the link or Twitter (#miCube) for the lastest information.
Fluorescence microscopy is an extremely powerful and versatile technique contributing to many areas of the life sciences. Especially variants featuring the ability to monitor single-molecule fluorescence, however, require sophisticated instrumentation that is either very expensive when bought commercially (>> 100 kEuro) or demands extensive expertise in optics and engineering.
Here we present an open and modular hardware framework aiming for
cost effectiveness: build your own starting at 20k Euro (~100 kEuro for state of the art capabilities)
modularity: all parts can be accessed and replaced by the user
simplicity: set up the microscope in a few hours without prior knowledge
customizability: confocal or widefield/TIRF microscopy,…
openness: part lists and drawings will be made available
stability and throughput: minimizing drift and utilise well plate scanners
Interested? Drop me a line. We are currently working together with several academic labs to bring their ideas to life and develop the miCube concept further.
S. Farooq and J. Hohlbein, Physical Chemistry Chemical Physics, 17, 27862, 2015, [link], open access
The achievable time resolution of camera-based single-molecule detection is often limited by the frame rate of the camera. Especially in experiments utilizing single-molecule Förster resonance energy transfer (smFRET) to probe conformational dynamics of biomolecules, increasing the frame rate by either pixel-binning or cropping the field of view decreases the number of molecules that can be monitored simultaneously. Here, we present a generalised excitation scheme termed stroboscopic alternating-laser excitation (sALEX) that significantly improves the time resolution without sacrificing highly parallelised detection in total internal reflection fluorescence (TIRF) microscopy. In addition, we adapt a technique known from diffusion-based confocal microscopy to analyse the complex shape of FRET efficiency histograms. We apply both sALEX and dynamic probability distribution analysis (dPDA) to resolve conformational dynamics of interconverting DNA hairpins in the millisecond time range.
G.W. Evans, J. Hohlbein, T. Craggs, L. Aigrain and A.N. Kapanidis, Nucleic Acids Research, 43, 5998-6008, 2015, [link], open access
DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary
nucleotide. We previously employed intraprotein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase–DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening
rate in ternary complexes with complementary nucleotide was 6 s^-1, much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non- complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.