Claus Seidel - Academia.edu (original) (raw)

Papers by Claus Seidel

The Journal of Physical Chemistry, 1996

Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives ... more Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a ...

Biophysical Journal, 2011

In a world where more people grow older aging-related neurodegeneration like Alzheimer's disease ... more In a world where more people grow older aging-related neurodegeneration like Alzheimer's disease (AD) affects more and more people. Today, AD can be diagnosed with certainty only post mortem, detecting insoluble b-amyloid peptide (Ab) aggregates and neurofibrillary tangles in the patient's brain tissue. Aggregates consisting of Ab are a fundamental pathologic feature of AD. Today in many studies, concentrations of monomeric Ab in body fluids are investigated, especially for diagnostic purposes. Nevertheless, for the detection, quantitation and qualification of aggregated pathologic Ab forms, also in the course of aging, a highly sensitive detection assay system for aggregated Ab species is necessary. We developed an ultra-sensitive assay for the detection of aggregated protein species out of body fluids. This highly specific and sensitive assay uses confocal fluorescence spectroscopy methods and is sensitive enough to detect single aggregates. For the procedure, pathologic aggregates out of body fluids are immobilized on a glass chip, subsequently fluorescence labeled and detected via confocal spectroscopy. Actually, we are optimizing the assay in concerns of instrumentation (imaging) and microscopy high-resolution and even super-resolution methods. We are developing methods to analyze aggregates via super-resolution microscopy. Setups like PAINT (Point Accumulation for Imaging in Nanoscale Topography) or STORM (Stochastic Optical Reconstruction Microscopy) allow resolutions in nanometer-range. PAINT is based on replacing the point-spreadfunction (PSF) of a fluorophore by a point in the middle of a 2D gaussian fit. First measurements show resolutions of 30 nm. STORM is based on highaccuracy localization of photoswitchable fluorophores. During one imaging cycle, only a small part of the fluorophores is turned on. This allows a high accuracy in determining the fluorophore position by replacing the PSF. The fluorophore positions obtained from a series of imaging cycles can be used to reconstruct the whole image. Spectral imaging techniques are important for many biological experiments, particularly live-cell quantitative imaging of multiple fluorescence probes. Existing hyperspectral imaging systems require sequential techniques, limiting the data acquisition rate. A newly-developed snapshot device, the Image Mapping Spectrometer (IMS), acquires full spectral information simultaneously from every pixel in the field with image acquisition rates up to 10 frames/second. The IMS maps adjacent pixels from the object to create space between them in the image, and then uses a grating to spread wavelength content from each pixel into this space. Direct image re-mapping provides the final 3D (x, y, lambda) data cube. Fluorescent protein (FP)-based biosensors are increasingly valuable tools for identifying subcellular dynamic processes in live cells. Many biosensors are based on FP-FRET, and measurements of the resultant small changes in FRET require high quality data. Tracking intracellular free Ca 2þ levels is also crucial to elucidating signaling events, but the best Ca 2þ indicator dyes overlap in the spectral emission range of the common FP-FRET biosensors. We have used the IMS system to simultaneously image multicolored FPs (i.e., CFP, GFP, YFP) in combination with extrinsic indicator dyes, such as Fluo-4. To demonstrate the speed and resolution of the IMS approach, we monitored both intracellular Ca 2þ oscillations and caspase-3 activity during hydrogen peroxide-induced apoptosis. Ca 2þ activity was measured with Fluo-4 (emission peak at 517 nm) and caspase-3 activity was measured with SCAT3.1, a FRET biosensor based on the ECFP (emission peak at 484 nm) and EYFP (emission peak at 527 nm) pair. Using the IMS, the three fluorophores were imaged with sub-second temporal resolution and spectrally unmixed in real-time. This permits direct correlation of Ca 2þ activity with other apoptotic signaling events and demonstrates the power of the IMS for measuring dynamic physiological processes. Diabetes is a disease resulting from changes in pancreatic islets, which are insulin secreting micro-organs within the pancreas. With increased blood glucose, insulin is secreted from beta cells in the islets in a coordinated pulsatile manner. At the same time alpha cell glucagon secretion is inhibited. Mechanisms controlling these processes at the intercellular and at the inter-islet level remain unclear. We suggest that the three-dimensional organization of islet cells and the dynamics of islet blood flow have a role in regulating insulin and glucagon secretion. This is suggested by observation that the density of blood vessels within islets is much greater than in surrounding pancreatic tissue, and that most individual islet cells are adjacent to a blood vessel. As an initial test of our hypothesis, we have developed a high-speed in vivo fluorescence imaging method to track pancreatic blood flow in a living mouse. We are also developing methods necessary to analyze the large amounts of data generated. Using high speed line scan confocal microscopy the method has full frame sub-micron spatial and less than 10 ms temporal resolution. Islets are located within the pancreas by using mice with GFP-labeled beta cells. Blood plasma is labeled with a fluorescent dextran, allowing mapping of vascular dimensions and pathway. Individual blood cells are fluorescently labeled by osmotic shock loading with an Alexa dye, which allows tracking of the blood flow. We present current results for blood flow under different levels of blood glucose in clamping experiments. Our previous qualitative results have suggested that there are differences in blood flow parameters at different glucose levels. Here, a more quantitative analysis of blood flow velocity, any observed changes in vessel dimensions, and changes in blood flow coverage inside and outside islets is presented. To study the structural organization of mitochondrial proteins, we applied Stimulated Emission Depletion (STED) microscopy in isolated mitochondria. In STED microscopy, two laser beams are used: one for excitation of fluorophores and the other, with doughnut shape, to deplete them in order to allow fluorescence emission only from the excited volume located at the doughnut's center. With STED a lateral resolution of~45 nm was achieved in images of isolated mitochondria. We investigated the localization pattern and distribution of MaxiKa, COX4 and VDAC1. After a combined analysis of classical confocal and STED images, we found distinct distributions for VDAC1, MaxiKa and COX4. COX4 distribution was consistent with localization in the cristae. We established that there are 7-15 clusters of Max-iKa, 10-15 clusters of VDAC1, and 15-20 clusters of COX4 per mitochondria. We have demonstrated that protein clusters in the mitochondria can be resolved with a separation power of~45 nm, and that it is possible to retrieve quantitative information about the number of clusters and density of proteins in mitochondria. This approach can be extended to eins in mitochondria and subcellular organelles. Supported by NIH.

Structure, 2015

Structures of biomolecular systems are increasingly computed by integrative modeling that relies ... more Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?

Nature communications, 2014

Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors.... more Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors. Understanding the molecular bases of their activation requires the characterization of the dynamic equilibrium between active and resting states. Here, we monitor, using single-molecule Förster resonance energy transfer, the kinetics of the reorientation of the extracellular ligand-binding domain of the metabotropic glutamate receptor (mGluR), a class C G-protein-coupled receptor. We demonstrate that most receptors oscillate between a resting- and an active-conformation on a sub-millisecond timescale. Interestingly, we demonstrate that differences in agonist efficacies stem from differing abilities to shift the conformational equilibrium towards the fully active state, rather than from the stabilization of alternative static conformations, which further highlights the dynamic nature of mGluRs and revises our understanding of receptor activation and allosteric modulation.

The Journal of Physical Chemistry B, 2007

We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluoresce... more We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluorescent dye, and micelles as membrane mimetic systems. In the presence of neutral micelles (Triton X-100 and Brij 35) R123 partitions between the aqueous solution and the micellar pseudo-phase, undergoing red shift of the absorption and the emission spectra. Fluorescence correlation spectroscopy (FCS) was used to study the dynamics of these systems over an extremely wide time range and at the single-molecule level, yielding information in one and the same experiment about the diffusional dynamics of free and bound rhodamine and about the dye-exchange dynamics as well as several photophysical properties of the rhodamine bound to the micelles. It was found that the entry rate constants are diffusion-controlled, indicating that there are no geometric or orientational requirements for the association of the dye with the micelle. With respect to the dye-exchange dynamics, micelles are found to behave as soft supramolecular cages in contrast to other rigid supramolecular cavities, such as cyclodextrins. The exit rate constants depend on the surfactant and determine the stability of the binding. Single-molecule multiparameter fluorescence detection (MFD) was used to examine the fluorescence properties of individual molecules in comparison to ensembles of molecules. The MFD histograms confirm the fast dye-exchange dynamics observed by FCS and yield mean values of fluorescence lifetimes and anisotropies in agreement with those obtained in bulk measurements.

The Journal of Physical Chemistry B, 1999

... Synthesis and Characterization of the Ground and Excited States of Tripodal-like Oligothienyl... more ... Synthesis and Characterization of the Ground and Excited States of Tripodal-like Oligothienyl-imidazoles. Jo o Pina, J. S rgio Seixas de Melo, Rosa MF Batista, Susana PG Costa and M. Manuela M. Raposo. The Journal of Physical Chemistry B 2010 114 (15), 4964-4972. ...

Proceedings of the National Academy of Sciences, 2003

Protein conformational transitions form the molecular basis of many cellular processes, such as s... more Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.

Proceedings of the National Academy of Sciences, 2009

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessi... more The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated ''Widom 601'' positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 Å measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.

Nature Structural & Molecular Biology, 2004

Synthesis of ATP from ADP and phosphate, catalyzed by F 0 F 1 -ATP synthases, is the most abundan... more Synthesis of ATP from ADP and phosphate, catalyzed by F 0 F 1 -ATP synthases, is the most abundant physiological reaction in almost any cell. F 0 F 1 -ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F 0 F 1 -ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The ␥ subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

Journal of the American Chemical Society, 2005

The control of supramolecular systems requires a thorough understanding of their dynamics on a mo... more The control of supramolecular systems requires a thorough understanding of their dynamics on a molecular level. We present fluorescence correlation spectroscopy (FCS) as a powerful spectroscopic tool to study supramolecular dynamics with single molecule sensitivity. The formation of a supramolecular complex between beta-cyclodextrin (beta-CD) as host and pyronines Y (PY) and B (PB) as guests is studied by FCS. Global target analysis of full correlation curves with a newly derived theoretical model yields in a single experiment the fluorescence lifetimes and the diffusion coefficients of free and complexed guests and the rate constants describing the complexation dynamics. These data give insight into the recently published surprising fact that the association equilibrium constant of beta-CD with PY is much lower than that with the much bulkier guest PB. FCS shows that the stability of the complexes is dictated by the dissociation and not by the association process. The association r...

Biophysical Journal, 2011

Stem cell number in shoot and floral meristems of Arabidopsis is regulated by the CLAVATA3 (CLV3)... more Stem cell number in shoot and floral meristems of Arabidopsis is regulated by the CLAVATA3 (CLV3) signalling pathway. Perception of the CLV3 peptide requires the receptor kinase CLAVATA1 (CLV1), the receptor-like protein CLAVATA2 (CLV2) and the kinase CORYNE (CRN). Genetic analysis suggested that CLV2 and CRN act together, and in parallel with CLV1. We studied the intracellular localization of receptor fusions with fluorescent protein tags, and their capacities for interaction via efficiency of fluorescence resonance energy transfer (E FRET ). We found that CLV2 and CRN require each other for export from the endoplasmatic reticulum (ER), and localization to the plasma membrane (PM). CRN readily forms homomers and interacts with CLV2 through the transmembrane domain and adjacent juxtamembrane sequences. CLV1 forms homomers independently of CLV2 and CRN at the PM. We propose that the CLV3 signal is perceived by a tetrameric CLV2/CRN complex and a CLV1 homodimer that localize to the PM and can interact via CRN. 2 www.plant.org on June 16, 2015 -Published by www.plantphysiol.org Downloaded from Bleckmann et al., CRN localizes CLV2 to the PM interact via their transmembrane domains, or immediately adjacent sequences (Fig. . A loss-of-function mutation of CRN, crn-1, is caused by an amino acid exchange within the predicted transmembrane domain, suggesting that membrane localization, interaction with a partner protein, or both is essential for CRN function.

Methods in enzymology, 2010

In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing importan... more In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Förster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters...

The Journal of Physical Chemistry B, 2010

Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to ... more Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to analyze conformational dynamics by Förster resonance energy transfer (FRET) measurements considering simulated and experimental data. First, an extension of photon distribution analysis (PDA) is applied to characterize conformational exchange between two or more states via global analysis of the shape of FRET peaks for different time bins. PDA accurately predicts the shape of FRET efficiency histograms in the presence of FRET fluctuations, taking into account shot noise and background contributions. Dynamic-PDA quantitatively recovers FRET efficiencies of the interconverting states and relaxation times of dynamics on the time scale of the diffusion time t d (typically milliseconds), with a dynamic range of the method of about (1 order of magnitude with respect to t d . Correction procedures are proposed to consider the factors limiting the accuracy of dynamic-PDA, such as brightness variations, shortening of the observation time due to diffusion, and a contribution of multimolecular events. Second, an analysis procedure for multiparameter fluorescence detection is presented, where intensity-derived FRET efficiency is correlated with the fluorescence lifetime of the donor quenched by FRET. If a maximum likelihood estimator is applied to compute a mean fluorescence lifetime of mixed states, one obtains a fluorescence weighted mean lifetime. Thus a mixed state is detected by a characteristic shift of the fluorescence lifetime, which becomes longer than that expected for a single species with the same intensity-derived FRET efficiency. Analysis tools for direct visual inspection of two-dimensional diagrams of FRET efficiency versus donor lifetime are presented for the cases of static and dynamic FRET. Finally these new techniques are compared with fluorescence correlation spectroscopy.

The Journal of Physical Chemistry B, 2008

Probability distribution analysis (PDA) [M. Antonik et al., J. Phys. Chem. B 2006, 110, 6970] all... more Probability distribution analysis (PDA) [M. Antonik et al., J. Phys. Chem. B 2006, 110, 6970] allows one to quantitatively analyze single-molecule (SM) data obtained in Förster resonance energy transfer (FRET) or fluorescence polarization experiments. By taking explicitly background and shot noise contributions into account, PDA accurately predicts the shape of one-dimensional histograms of various parameters, such as FRET efficiency or fluorescence anisotropy. In order to describe complex experimental SM-FRET or polarization data obtained for systems consisting of multiple non-interconverting fluorescent states, several extensions to the PDA theory are presented. Effects of brightness variations and multiple-molecule events are considered independently of the detection volume parameters by using only the overall experimental signal intensity distribution. The extended PDA theory can now be applied to analyze any mixture, by using any a priori model or a model-free deconvolution approach based on the maximum entropy method (MEM). The accuracy of the analysis and the number of free parameters are limited only by data quality. Correction of the PDA model function for the presence of multiple-molecule events allows one to measure at high SM concentrations to avoid artifacts due to a very long measurement time. Tools such as MEM and combined mean donor fluorescence lifetime analysis have been developed to distinguish whether extra broadening of PDA histograms could be attributed to structural heterogeneities or dye artifacts. In this way, an ultimate resolution in FRET experiments in the range of a few Ångström is achieved which allows for molecular Ångström optics distinguishing between a set of fixed distances and a distribution of distances. The extended theory is verified by analyzing simulations and experimental data.

The Journal of Physical Chemistry A, 1999

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel dete... more Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time ...

The Journal of Physical Chemistry A, 1998

The Journal of Physical Chemistry, 1996

Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives ... more Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a ...

Biophysical Journal, 2011

In a world where more people grow older aging-related neurodegeneration like Alzheimer's disease ... more In a world where more people grow older aging-related neurodegeneration like Alzheimer's disease (AD) affects more and more people. Today, AD can be diagnosed with certainty only post mortem, detecting insoluble b-amyloid peptide (Ab) aggregates and neurofibrillary tangles in the patient's brain tissue. Aggregates consisting of Ab are a fundamental pathologic feature of AD. Today in many studies, concentrations of monomeric Ab in body fluids are investigated, especially for diagnostic purposes. Nevertheless, for the detection, quantitation and qualification of aggregated pathologic Ab forms, also in the course of aging, a highly sensitive detection assay system for aggregated Ab species is necessary. We developed an ultra-sensitive assay for the detection of aggregated protein species out of body fluids. This highly specific and sensitive assay uses confocal fluorescence spectroscopy methods and is sensitive enough to detect single aggregates. For the procedure, pathologic aggregates out of body fluids are immobilized on a glass chip, subsequently fluorescence labeled and detected via confocal spectroscopy. Actually, we are optimizing the assay in concerns of instrumentation (imaging) and microscopy high-resolution and even super-resolution methods. We are developing methods to analyze aggregates via super-resolution microscopy. Setups like PAINT (Point Accumulation for Imaging in Nanoscale Topography) or STORM (Stochastic Optical Reconstruction Microscopy) allow resolutions in nanometer-range. PAINT is based on replacing the point-spreadfunction (PSF) of a fluorophore by a point in the middle of a 2D gaussian fit. First measurements show resolutions of 30 nm. STORM is based on highaccuracy localization of photoswitchable fluorophores. During one imaging cycle, only a small part of the fluorophores is turned on. This allows a high accuracy in determining the fluorophore position by replacing the PSF. The fluorophore positions obtained from a series of imaging cycles can be used to reconstruct the whole image. Spectral imaging techniques are important for many biological experiments, particularly live-cell quantitative imaging of multiple fluorescence probes. Existing hyperspectral imaging systems require sequential techniques, limiting the data acquisition rate. A newly-developed snapshot device, the Image Mapping Spectrometer (IMS), acquires full spectral information simultaneously from every pixel in the field with image acquisition rates up to 10 frames/second. The IMS maps adjacent pixels from the object to create space between them in the image, and then uses a grating to spread wavelength content from each pixel into this space. Direct image re-mapping provides the final 3D (x, y, lambda) data cube. Fluorescent protein (FP)-based biosensors are increasingly valuable tools for identifying subcellular dynamic processes in live cells. Many biosensors are based on FP-FRET, and measurements of the resultant small changes in FRET require high quality data. Tracking intracellular free Ca 2þ levels is also crucial to elucidating signaling events, but the best Ca 2þ indicator dyes overlap in the spectral emission range of the common FP-FRET biosensors. We have used the IMS system to simultaneously image multicolored FPs (i.e., CFP, GFP, YFP) in combination with extrinsic indicator dyes, such as Fluo-4. To demonstrate the speed and resolution of the IMS approach, we monitored both intracellular Ca 2þ oscillations and caspase-3 activity during hydrogen peroxide-induced apoptosis. Ca 2þ activity was measured with Fluo-4 (emission peak at 517 nm) and caspase-3 activity was measured with SCAT3.1, a FRET biosensor based on the ECFP (emission peak at 484 nm) and EYFP (emission peak at 527 nm) pair. Using the IMS, the three fluorophores were imaged with sub-second temporal resolution and spectrally unmixed in real-time. This permits direct correlation of Ca 2þ activity with other apoptotic signaling events and demonstrates the power of the IMS for measuring dynamic physiological processes. Diabetes is a disease resulting from changes in pancreatic islets, which are insulin secreting micro-organs within the pancreas. With increased blood glucose, insulin is secreted from beta cells in the islets in a coordinated pulsatile manner. At the same time alpha cell glucagon secretion is inhibited. Mechanisms controlling these processes at the intercellular and at the inter-islet level remain unclear. We suggest that the three-dimensional organization of islet cells and the dynamics of islet blood flow have a role in regulating insulin and glucagon secretion. This is suggested by observation that the density of blood vessels within islets is much greater than in surrounding pancreatic tissue, and that most individual islet cells are adjacent to a blood vessel. As an initial test of our hypothesis, we have developed a high-speed in vivo fluorescence imaging method to track pancreatic blood flow in a living mouse. We are also developing methods necessary to analyze the large amounts of data generated. Using high speed line scan confocal microscopy the method has full frame sub-micron spatial and less than 10 ms temporal resolution. Islets are located within the pancreas by using mice with GFP-labeled beta cells. Blood plasma is labeled with a fluorescent dextran, allowing mapping of vascular dimensions and pathway. Individual blood cells are fluorescently labeled by osmotic shock loading with an Alexa dye, which allows tracking of the blood flow. We present current results for blood flow under different levels of blood glucose in clamping experiments. Our previous qualitative results have suggested that there are differences in blood flow parameters at different glucose levels. Here, a more quantitative analysis of blood flow velocity, any observed changes in vessel dimensions, and changes in blood flow coverage inside and outside islets is presented. To study the structural organization of mitochondrial proteins, we applied Stimulated Emission Depletion (STED) microscopy in isolated mitochondria. In STED microscopy, two laser beams are used: one for excitation of fluorophores and the other, with doughnut shape, to deplete them in order to allow fluorescence emission only from the excited volume located at the doughnut's center. With STED a lateral resolution of~45 nm was achieved in images of isolated mitochondria. We investigated the localization pattern and distribution of MaxiKa, COX4 and VDAC1. After a combined analysis of classical confocal and STED images, we found distinct distributions for VDAC1, MaxiKa and COX4. COX4 distribution was consistent with localization in the cristae. We established that there are 7-15 clusters of Max-iKa, 10-15 clusters of VDAC1, and 15-20 clusters of COX4 per mitochondria. We have demonstrated that protein clusters in the mitochondria can be resolved with a separation power of~45 nm, and that it is possible to retrieve quantitative information about the number of clusters and density of proteins in mitochondria. This approach can be extended to eins in mitochondria and subcellular organelles. Supported by NIH.

Structure, 2015

Structures of biomolecular systems are increasingly computed by integrative modeling that relies ... more Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?

Nature communications, 2014

Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors.... more Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors. Understanding the molecular bases of their activation requires the characterization of the dynamic equilibrium between active and resting states. Here, we monitor, using single-molecule Förster resonance energy transfer, the kinetics of the reorientation of the extracellular ligand-binding domain of the metabotropic glutamate receptor (mGluR), a class C G-protein-coupled receptor. We demonstrate that most receptors oscillate between a resting- and an active-conformation on a sub-millisecond timescale. Interestingly, we demonstrate that differences in agonist efficacies stem from differing abilities to shift the conformational equilibrium towards the fully active state, rather than from the stabilization of alternative static conformations, which further highlights the dynamic nature of mGluRs and revises our understanding of receptor activation and allosteric modulation.

The Journal of Physical Chemistry B, 2007

We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluoresce... more We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluorescent dye, and micelles as membrane mimetic systems. In the presence of neutral micelles (Triton X-100 and Brij 35) R123 partitions between the aqueous solution and the micellar pseudo-phase, undergoing red shift of the absorption and the emission spectra. Fluorescence correlation spectroscopy (FCS) was used to study the dynamics of these systems over an extremely wide time range and at the single-molecule level, yielding information in one and the same experiment about the diffusional dynamics of free and bound rhodamine and about the dye-exchange dynamics as well as several photophysical properties of the rhodamine bound to the micelles. It was found that the entry rate constants are diffusion-controlled, indicating that there are no geometric or orientational requirements for the association of the dye with the micelle. With respect to the dye-exchange dynamics, micelles are found to behave as soft supramolecular cages in contrast to other rigid supramolecular cavities, such as cyclodextrins. The exit rate constants depend on the surfactant and determine the stability of the binding. Single-molecule multiparameter fluorescence detection (MFD) was used to examine the fluorescence properties of individual molecules in comparison to ensembles of molecules. The MFD histograms confirm the fast dye-exchange dynamics observed by FCS and yield mean values of fluorescence lifetimes and anisotropies in agreement with those obtained in bulk measurements.

The Journal of Physical Chemistry B, 1999

... Synthesis and Characterization of the Ground and Excited States of Tripodal-like Oligothienyl... more ... Synthesis and Characterization of the Ground and Excited States of Tripodal-like Oligothienyl-imidazoles. Jo o Pina, J. S rgio Seixas de Melo, Rosa MF Batista, Susana PG Costa and M. Manuela M. Raposo. The Journal of Physical Chemistry B 2010 114 (15), 4964-4972. ...

Proceedings of the National Academy of Sciences, 2003

Protein conformational transitions form the molecular basis of many cellular processes, such as s... more Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.

Proceedings of the National Academy of Sciences, 2009

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessi... more The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated ''Widom 601'' positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 Å measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.

Nature Structural & Molecular Biology, 2004

Synthesis of ATP from ADP and phosphate, catalyzed by F 0 F 1 -ATP synthases, is the most abundan... more Synthesis of ATP from ADP and phosphate, catalyzed by F 0 F 1 -ATP synthases, is the most abundant physiological reaction in almost any cell. F 0 F 1 -ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F 0 F 1 -ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The ␥ subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

Journal of the American Chemical Society, 2005

The control of supramolecular systems requires a thorough understanding of their dynamics on a mo... more The control of supramolecular systems requires a thorough understanding of their dynamics on a molecular level. We present fluorescence correlation spectroscopy (FCS) as a powerful spectroscopic tool to study supramolecular dynamics with single molecule sensitivity. The formation of a supramolecular complex between beta-cyclodextrin (beta-CD) as host and pyronines Y (PY) and B (PB) as guests is studied by FCS. Global target analysis of full correlation curves with a newly derived theoretical model yields in a single experiment the fluorescence lifetimes and the diffusion coefficients of free and complexed guests and the rate constants describing the complexation dynamics. These data give insight into the recently published surprising fact that the association equilibrium constant of beta-CD with PY is much lower than that with the much bulkier guest PB. FCS shows that the stability of the complexes is dictated by the dissociation and not by the association process. The association r...

Biophysical Journal, 2011

Stem cell number in shoot and floral meristems of Arabidopsis is regulated by the CLAVATA3 (CLV3)... more Stem cell number in shoot and floral meristems of Arabidopsis is regulated by the CLAVATA3 (CLV3) signalling pathway. Perception of the CLV3 peptide requires the receptor kinase CLAVATA1 (CLV1), the receptor-like protein CLAVATA2 (CLV2) and the kinase CORYNE (CRN). Genetic analysis suggested that CLV2 and CRN act together, and in parallel with CLV1. We studied the intracellular localization of receptor fusions with fluorescent protein tags, and their capacities for interaction via efficiency of fluorescence resonance energy transfer (E FRET ). We found that CLV2 and CRN require each other for export from the endoplasmatic reticulum (ER), and localization to the plasma membrane (PM). CRN readily forms homomers and interacts with CLV2 through the transmembrane domain and adjacent juxtamembrane sequences. CLV1 forms homomers independently of CLV2 and CRN at the PM. We propose that the CLV3 signal is perceived by a tetrameric CLV2/CRN complex and a CLV1 homodimer that localize to the PM and can interact via CRN. 2 www.plant.org on June 16, 2015 -Published by www.plantphysiol.org Downloaded from Bleckmann et al., CRN localizes CLV2 to the PM interact via their transmembrane domains, or immediately adjacent sequences (Fig. . A loss-of-function mutation of CRN, crn-1, is caused by an amino acid exchange within the predicted transmembrane domain, suggesting that membrane localization, interaction with a partner protein, or both is essential for CRN function.

Methods in enzymology, 2010

In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing importan... more In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Förster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters...

The Journal of Physical Chemistry B, 2010

Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to ... more Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to analyze conformational dynamics by Förster resonance energy transfer (FRET) measurements considering simulated and experimental data. First, an extension of photon distribution analysis (PDA) is applied to characterize conformational exchange between two or more states via global analysis of the shape of FRET peaks for different time bins. PDA accurately predicts the shape of FRET efficiency histograms in the presence of FRET fluctuations, taking into account shot noise and background contributions. Dynamic-PDA quantitatively recovers FRET efficiencies of the interconverting states and relaxation times of dynamics on the time scale of the diffusion time t d (typically milliseconds), with a dynamic range of the method of about (1 order of magnitude with respect to t d . Correction procedures are proposed to consider the factors limiting the accuracy of dynamic-PDA, such as brightness variations, shortening of the observation time due to diffusion, and a contribution of multimolecular events. Second, an analysis procedure for multiparameter fluorescence detection is presented, where intensity-derived FRET efficiency is correlated with the fluorescence lifetime of the donor quenched by FRET. If a maximum likelihood estimator is applied to compute a mean fluorescence lifetime of mixed states, one obtains a fluorescence weighted mean lifetime. Thus a mixed state is detected by a characteristic shift of the fluorescence lifetime, which becomes longer than that expected for a single species with the same intensity-derived FRET efficiency. Analysis tools for direct visual inspection of two-dimensional diagrams of FRET efficiency versus donor lifetime are presented for the cases of static and dynamic FRET. Finally these new techniques are compared with fluorescence correlation spectroscopy.

The Journal of Physical Chemistry B, 2008

Probability distribution analysis (PDA) [M. Antonik et al., J. Phys. Chem. B 2006, 110, 6970] all... more Probability distribution analysis (PDA) [M. Antonik et al., J. Phys. Chem. B 2006, 110, 6970] allows one to quantitatively analyze single-molecule (SM) data obtained in Förster resonance energy transfer (FRET) or fluorescence polarization experiments. By taking explicitly background and shot noise contributions into account, PDA accurately predicts the shape of one-dimensional histograms of various parameters, such as FRET efficiency or fluorescence anisotropy. In order to describe complex experimental SM-FRET or polarization data obtained for systems consisting of multiple non-interconverting fluorescent states, several extensions to the PDA theory are presented. Effects of brightness variations and multiple-molecule events are considered independently of the detection volume parameters by using only the overall experimental signal intensity distribution. The extended PDA theory can now be applied to analyze any mixture, by using any a priori model or a model-free deconvolution approach based on the maximum entropy method (MEM). The accuracy of the analysis and the number of free parameters are limited only by data quality. Correction of the PDA model function for the presence of multiple-molecule events allows one to measure at high SM concentrations to avoid artifacts due to a very long measurement time. Tools such as MEM and combined mean donor fluorescence lifetime analysis have been developed to distinguish whether extra broadening of PDA histograms could be attributed to structural heterogeneities or dye artifacts. In this way, an ultimate resolution in FRET experiments in the range of a few Ångström is achieved which allows for molecular Ångström optics distinguishing between a set of fixed distances and a distribution of distances. The extended theory is verified by analyzing simulations and experimental data.

The Journal of Physical Chemistry A, 1999

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel dete... more Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time ...

The Journal of Physical Chemistry A, 1998