A5044593942- Protein Structure and Dynamics
- Advanced Fluorescence Microscopy Techniques
- Enzyme Structure and Function
- Quantum, superfluid, helium dynamics
- Spectroscopy and Quantum Chemical Studies
- RNA and protein synthesis mechanisms
- Force Microscopy Techniques and Applications
- Atomic and Subatomic Physics Research
- RNA Research and Splicing
- Mass Spectrometry Techniques and Applications
- Molecular Junctions and Nanostructures
- Bacteriophages and microbial interactions
- DNA and Nucleic Acid Chemistry
- Cold Atom Physics and Bose-Einstein Condensates
- Hemoglobin structure and function
- Photosynthetic Processes and Mechanisms
- Advanced biosensing and bioanalysis techniques
- Advanced Electron Microscopy Techniques and Applications
- Heat shock proteins research
- Microfluidic and Capillary Electrophoresis Applications
- Analytical Chemistry and Chromatography
- Advanced NMR Techniques and Applications
- Innovative Microfluidic and Catalytic Techniques Innovation
- RNA Interference and Gene Delivery
- Monoclonal and Polyclonal Antibodies Research
University of Zurich
2016-2025
University of Maryland, College Park
2010
University of Fribourg
2000-2007
University of Bonn
2002
Many eukaryotic proteins are disordered under physiological conditions, and fold into ordered structures only on binding to their cellular targets. Such intrinsically (IDPs) often contain a large fraction of charged amino acids. Here, we use single-molecule Förster resonance energy transfer investigate the influence residues dimensions unfolded proteins. We find that, in contrast compact conformations that have been observed for many at low denaturant concentration, IDPs can exhibit...
The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition solution conditions, especially salt denaturant concentration. However, the quantitative implications this behavior have remained unclear, largely because effective theta-state, central reference point for underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy two-focus correlation to determine...
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro vivo. However, generalized protocols FRET standards ensure the reproducibility accuracy measurements efficiencies are currently lacking. Here we report results a comparative blind study which 20 labs determined (E) several dye-labeled DNA duplexes. Using unified, straightforward method, obtained with s.d. between ±0.02 ±0.05. We...
We use the statistics of photon emission from single molecules to probe ultrafast dynamics an unfolded protein via Förster resonance energy transfer. Global reconfiguration chain occurs on a time scale ≈50 ns and slows down concomitant with collapse under folding conditions. These diffusive provide missing link between phenomenological chemical kinetics commonly used in physical description terms quantitative free surfaces. The experiments demonstrate potential single-molecule methods...
Internal friction, which reflects the "roughness" of energy landscape, plays an important role for proteins by modulating dynamics their folding and other conformational changes. However, experimental quantification internal friction its contribution to has remained challenging. Here we use combination single-molecule Förster resonance transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing determine reconfiguration times unfolded investigate mechanisms contributing...
Significance In the interior of a cell, volume accessible to each protein molecule is restricted by presence large number other macromolecules. Such crowded environment known affect stability and folding rates proteins. case intrinsically disordered proteins (IDPs), however, class that lack stable structure, much less about role crowding effects. We have quantified conformational changes occurring in IDPs high concentrations different polymers act as agents. Using single-molecule...
There has been a long-standing controversy regarding the effect of chemical denaturants on dimensions unfolded and intrinsically disordered proteins: A wide range experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no such increase radius gyration (Rg). This inconsistency challenges our current understanding mechanism denaturants, which are widely employed to...
We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on dimensions unfolded proteins. With FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us probe a wide range denaturant concentrations temperatures. find compaction state small cold shock protein increasing both presence absence denaturant, good agreement between results from dynamic...
We have used the combination of single-molecule Förster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe conformational ensemble collapsed unfolded state small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult access experimentally, because equilibrium signal in dominated by folded molecules. Here, we avoid this problem two ways. One use transfer, which allows separation...
Significance A large number of naturally occurring proteins are now known to be unstructured under physiological conditions. Many these intrinsically disordered (IDPs) bind other biological macromolecules or ligands and involved in important regulatory processes the cell. For understanding structural basis functional properties, it is essential quantify balance interactions that modulate heterogeneous conformational distributions IDPs unfolded general. In contrast behavior expected for...
Single-molecule fluorescence spectroscopy and correlation methods are finding increasing applications in the investigation of biomolecular dynamics, especially together with Förster resonance energy transfer (FRET). Here, we use combination start−stop experiments classical (FCS) to obtain complete intensity auto- cross-correlation functions from picoseconds seconds for investigating dynamics unfolded proteins peptides. In distance information single-molecule efficiency histograms, can...
The properties of unfolded proteins are essential both for the mechanisms protein folding and function large group intrinsically disordered proteins. However, detailed structural dynamical characterization these highly dynamic conformationally heterogeneous ensembles has remained challenging. Here we combine compare three leading techniques investigation proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), single-molecule Förster resonance energy transfer (FRET), with goal...
Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics protein folding. Recent experiments on spectrin domains provided first evidence for a pronounced contribution proteins that fold millisecond timescale. However, it has remained unclear how this is distributed along reaction what influence folding dynamics. Here we use combination single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy,...
Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration posttranslational modifications. However, little is known about the effect of macromolecular crowding on kinetics stability IDPs with their targets. Here, we investigate influence coupled folding binding between two IDPs, using polyethylene glycol as a agent across broad size range. Single-molecule F\"orster resonance energy transfer...
Single molecule Förster resonance energy transfer (FRET) experiments are a versatile method for investigating the conformational distributions and dynamics of biological macromolecules. In common type experiment, fluorescence bursts from individual molecules freely diffusing in solution detected as they pass through observation volume confocal microscope. Correlation analysis shows that under typical experimental conditions, time scales up to several tens milliseconds, probability return is...
Significance Molecular chaperones are a group of proteins that essential for avoiding the aggregation other in crowded cellular environment. Chaperones function by interacting with these substrate different ways. However, it has remained challenge to measure changes occur and understand how prevent misfolding or aggregation. Here we investigate chaperone system keeps protein denatured clamping polypeptide chain. We observe an expansion chain up 30-fold volume owing steric repulsion between...
Abstract Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments molecular simulations investigate how between immunoglobulin-like titin is prevented. Surprisingly, find that during refolding repeats, independent sequence...
Significance The dynamics of proteins, which are essential for both folding and function, known to be strongly dependent on solvent viscosity friction. However, an increasing number experiments have demonstrated the importance a contribution protein independent Such “internal friction” has recently been detected even in unfolded although they more expanded solvent-accessible than folded proteins. Based two complementary experimental methods, simulations, theory, our results provide coherent...