Jaroslav Večeř

Charles University

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Petr Heřman

Charles University

Tomáš Obšil

Czech Academy of Sciences, Institute of Physiology

Veronika Obšilová

Czech Academy of Sciences

Karel Sigler

Czech Academy of Sciences, Institute of Microbiology

MŠ

Miroslav Šulc

Charles University

Jaromı́r Plášek

Czech Academy of Sciences, Institute of Physics

Jan Teisinger

Czech Academy of Sciences, Institute of Physiology

J Malínský

Czech Academy of Sciences

Dana Gášková

Charles University

Aleš Holoubek

Institute of Haematology and Blood Transfusion

Cooperative Institutions

Charles University

Czech Academy of Sciences

Czech Academy of Sciences, Institute of Physics

Czech Academy of Sciences, Institute of Microbiology

Czech Academy of Sciences, Institute of Physiology

Institute of Protein Biochemistry

Centre National de la Recherche Scientifique

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

National Research Council

Institute of Food Science

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LOW‐TEMPERATURE LUMINESCENCE SPECTRA AND FLUORESCENCE LIFETIMES OF POLYCYTIDYLIC ACID IN POLYALCOHOLIC GLASSES

Photochemistry and Photobiology (1993)

Jaroslav Večeř Petr Heřman Josef Beránek

Abstract— Corrected emission spectra and fluorescence lifetimes of polycytidylic acid in ethylene glycol: water glass at low temperatures are reported. Luminescence properties observed exhibit a strong dependence on pH and temperature. At neutral pH a vibronic structure of a blue part of the fluorescence spectrum is revealed when temperature is changed from 77 to 10 K, confirming that a monomer component of fluorescence is present. There is also a strong difference in decay of a red‐shifted excimer fluorescence at 10 K at pH 7 and pH 3.9, reflecting a different protonation of cytosine residues and different conformations of polynucleotides in such conditions.

Excimer

Polynucleotide

10.1111/j.1751-1097.1993.tb09212.x

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Citations (1)

The 14-3-3 Protein Affects the Conformation of the Regulatory Domain of Human Tyrosine Hydroxylase

Biochemistry (2008)

Veronika Obšilová Eliska Nedbalkova Jan Šilhán Evžen Bouřa Petr Heřman

Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kinases. Phosphorylation of Ser19 does not by itself increase TH activity but induces its binding to the 14-3-3 protein. That leads to the enhancement of TH activity with a still not fully understood mechanism. The main goal of this work was to investigate whether the 14-3-3 protein binding affects the conformation of the regulatory domain of human TH isoform 1 (TH1R). Site-directed mutagenesis was used to generate five single-tryptophan mutants of TH1R with the Trp residue located at five different positions within the domain (positions 14, 34, 73, 103, and 131). Time-resolved tryptophan fluorescence measurements revealed that phosphorylation of Ser19 and Ser40 does not itself induce any significant structural changes in regions surrounding inserted tryptophans. On the other hand, the interaction between the 14-3-3 protein and phosphorylated TH1R decreases the solvent exposure of tryptophan residues at positions 14 and 34 and induces distinct structural change in the vicinity of Trp73. The 14-3-3 protein binding also reduces the sensitivity of phosphorylated TH1R to proteolysis by protecting its N-terminal part (first 33 residues). Circular dichroism measurements showed that TH1R is an unstructured protein with a low content of secondary structure and that neither phosphorylation nor the 14-3-3 protein binding changes its secondary structure.

Tryptophan hydroxylase

Proteolysis

10.1021/bi7019468

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Citations (50)

Polarized phosphorescence of adeninium hemisulphate hydrate single crystal

Journal of Luminescence (1990)

Jaroslav Večeř Vladimı́r Baumruk O. Jelínek

Crystal (programming language)

Clathrate hydrate

10.1016/0022-2313(90)90004-u

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Citations (0)

Excimer emission of molecular systems with energy transfer

The Journal of Chemical Physics (1990)

Jaroslav Večeř

The exact solution of kinetic equations of a simple chain of molecular excimer forming system is derived. The influence of energy transfer on both monomer and excimer emission kinetic is discussed.

Excimer

10.1063/1.458692

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Short-lived fluorescence component of DPH reports on lipid–water interface of biological membranes

Chemistry and Physics of Lipids (2004)

Ivo Konopásek Jaroslav Večeř Kazimierz Strzałka Evžen Amler

10.1016/j.chemphyslip.2004.02.005

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Citations (21)

Time-Resolved Fluorescence in Micellar Systems: A Critical Application to the Partitioning of Naphthalene in Aqueous Sodium Dodecyl Sulfate

Langmuir (1996)

Andrzej Kowalczyk Jaroslav Večeř Brian Hodgson J. P. Keene Robert E. Dale

Static and dynamic aspects of the partitioning of naphthalene into the hydrocarbon phase in aqueous sodium dodecyl sulfate micelles have been critically examined by nanosecond time-resolved pulse fluorometry. It is shown that, in such a system, a properly detailed analysis of the excited-state decay behavior as a function of progressive partitioning of the solute into increasing concentrations of micelles is capable of revealing and directly quantitating the underlying kinetics of the reversible transfer of naphthalene in its excited state between aqueous and micellar phases. The time-resolved data indicate that, in the aqueous sodium dodecyl sulfate system, the partition coefficient for excited-state naphthalene can differ only marginally from that for the ground state. From the estimate obtained for the rate coefficient for entry of excited-state naphthalene into micelles, it would appear that any barrier to the crossing of excited naphthalene into the micelle, given that a collision has taken place, is likely to be rather small. The contrasting time-resolved behavior of a system in which the basis lifetime of the fluorescence probe in the micellar environment is shorter than that in the bulk environment rather than longer, as in the case examined experimentally, is also modeled. The effects of quenching of the fluorescence at the interface between the micellar and aqueous phases are also examined and discussed in the context of the aqueous cetyltrimethylammonium bromide system.

Sodium dodecyl sulfate

10.1021/la9602729

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Temperature modulates binding specificity and affinity of the d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics (2007)

Petr Heřman Ivan Barvı́k Maria Staiano Annalisa Vitale Jaroslav Večeř

Maltose-binding protein

Thermococcus

Hyperthermophile

10.1016/j.bbapap.2007.03.005

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Citations (13)

A Recombinant Glutamine-Binding Protein from Escherichia coli: Effect of Ligand-Binding on Protein Conformational Dynamics

Biotechnology Progress (2004)

Petr Heřman Jaroslav Večeř Viviana Scognamiglio Maria Staiano Mauro Rossi

We have investigated the effect of the binding of glutamine on the conformational dynamics of the recombinant glutamine binding protein (GlnBP) from Escherichia coli by steady-state and time-resolved fluorescence techniques. The structural stability of the protein was also studied by far-UV circular dichroism spectroscopy in the range of temperature between 25 and 80 °C. The results showed that the interaction of the protein with the ligand resulted in a marked change of the structural and conformational dynamics features of the protein. In particular, the fluorescence and circular dichroism data showed that the presence of glutamine resulted in a dramatic increase of the protein thermal stability of about 10 °C. In addition, the fluorescence time-resolved data pointed out that both in the absence and in the presence of glutamine the protein structure was highly rigid with small amplitude of segmental motion up to 65 °C and a low accessibility of the protein tryptophan residues to acrylamide. The obtained results on the structural properties of the recombinant glutamine-binding protein in the absence and in the presence of glutamine can contribute to a better understanding of the transport-related functions of the protein and structurally similar periplasmic transport proteins, as well as to the design and development of new biotechnological applications of this class of proteins.

Dynamics

Conformational change

10.1021/bp049956u

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Citations (10)

The C-Terminal Segment of Yeast BMH Proteins Exhibits Different Structure Compared to Other 14-3-3 Protein Isoforms

Biochemistry (2010)

Dana Veisova Lenka Řežábková Miroslav Štěpánek Pavlína Novotná Petr Heřman

Yeast 14-3-3 protein isoforms BMH1 and BMH2 possess a distinctly variant C-terminal tail which differentiates them from the isoforms of higher eukaryotes. Their C-termini are longer and contain a polyglutamine stretch of unknown function. It is now well established that the C-terminal segment of 14-3-3 proteins plays an important regulatory role by functioning as an autoinhibitor which occupies the ligand binding groove and blocks the binding of inappropriate ligands. Whether the same holds true or not for the yeast isoforms is unclear. Therefore, we investigated the conformational behavior of the C-terminal segment of BMH proteins using various biophysical techniques. Dynamic light scattering, sedimentation velocity, time-resolved fluorescence anisotropy decay, and size exclusion chromatography measurements showed that the molecules of BMH proteins are significantly larger compared to the human 14-3-3zeta isoform. On the other hand, the sedimentation analysis confirmed that BMH proteins form dimers. Time-resolved tryptophan fluorescence experiments revealed no dramatic structural changes of the C-terminal segment upon the ligand binding. Taken together, the C-terminal segment of BMH proteins adopts a widely opened and extended conformation that makes difficult its folding into the ligand binding groove, thus increasing the apparent molecular size. It seems, therefore, that the C-terminal segment of BMH proteins does not function as an autoinhibitor.

10.1021/bi100273k

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Citations (28)

14-3-3 Protein Interacts with Nuclear Localization Sequence of Forkhead Transcription Factor FoxO4

Biochemistry (2005)

Veronika Obšilová Jaroslav Večeř Petr Heřman Anna Pabianova Miroslav Šulc

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner. 14-3-3 proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of nuclear localization sequence (NLS). Masking or obscuring of NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equilibrium of FoxO localization toward the cytoplasm. According to our best knowledge, there is no experimental evidence showing a direct interaction between the 14-3-3 protein and NLS of FoxO. Therefore, the main goal of this work was to investigate whether the phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of FoxO4 NLS. We have used site-directed labeling of FoxO4 NLS with the extrinsic fluorophore 1,5-IAEDANS in conjunction with steady-state and time-resolved fluorescence spectroscopy to study conformational changes of FoxO4 NLS in vitro. Our data show that the 14-3-3 protein binding significantly changes the environment around AEDANS-labeled NLS and reduces its flexibility. On the other hand, the phosphorylation itself and the binding of double-stranded DNA have a small effect on the structure of this region. Our results also suggest that the DNA-binding domain of FoxO4 remains relatively mobile while bound to the 14-3-3 protein.

NLS

Forkhead Transcription Factors

10.1021/bi050618r

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Citations (113)