Tomas Fessl

University of South Bohemia in České Budějovice · Institute of Chemistry and Biochemistry (UCH)

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein co...

The Sec translocon is a transmembrane assembly highly conserved among all forms of life as the principal route for transport of polypeptides across or into lipid bilayers. In bacteria translocation involves allosteric communication between the membrane pore SecYEG and the associated SecA ATPase. Using singlemolecule fluorescence we reveal that slow...

Studies on DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA–ligand interactions in-side the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated wit...

High-resolution studies of DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this issue, we developed an in-cell NMR-based approach for monitoring DNA–ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of high-resolution NMR da...

Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the direct...

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques...

Primary single molecule fluorescence data file for use with the Python script.

Python script source code.

Conventional biophysical and chemical biology approaches for delineating relationships between the structure and biological function of nucleic acids (NAs) abstract NAs from their native biological context. However, cumulative experimental observations have revealed that the structure, dynamics and interactions of NAs might be strongly influenced b...

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon – SecY MK EG – resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolys...

C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, a direct experimental evidence for the existence of these structures in vivo has been missing. Whether i-motif structures form in complex environment of...

C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, a direct experimental evidence for the existence of these structures in vivo has been missing. Whether i-motif structures form in complex environment of...

Chromatin-remodeling ATPases modulate histones-DNA interactions within nucleosomes and regulate transcription. At the heart of remodeling, ATPase is a helicase-like motor flanked by a variety of conserved targeting domains. CHD4 is the core subunit of the nucleosome remodeling and deacetylase complex NuRD and harbors tandem plant homeo finger (tPHD...

Indocarbocyanine fluorophores attached via the 5' terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix l...

The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection...

Fluorescence resonance energy transfer (FRET) under in vivo conditions is a well-established technique for the evaluation of populations of protein bound/unbound nucleic acid (NA) molecules or NA hybridization kinetics. However, in vivo FRET has not been applied to in vivo quantitative conformational analysis of NA thus far. Here we explored parame...

Molecular dynamics (MD) simulations and ab initio quantum chemical calculations were employed to investigate the structure, dynamics and interactions of the QSY 21 nonfluorescent quencher and the fluorescence dye Rhodamine 6G bound to a B-DNA decamer. For QSY 21, two binding motifs were observed. In the first motif, the central xanthene ring is sta...

Molecular dynamics (MD) simulation and ab initio quantum mechanical calculations were employed to investigate the structure and dynamics of binding of QSY 21 nonfluorescent quencher to B-DNA decamer. Two binding motifs were observed: 1. Central xanthone ring of QSY 21 dye is oriented paralelly with the nearest DNA base pair and the complex is stabi...

a b s t r a c t Imaging of small objects such as single molecules, DNA clusters and single bacterial cells is problematic not only due to the lateral resolution that is obtainable in currently existing microscopy but also, and as much fundamentally limiting, due to the lack of sufficient axial depth of focus to have the full object focused simultan...