Topics

The research of the Hof Fluorescence Group might be defined by three research directions:


Project 1:

Understanding Membrane Biophysics on an Atomistic Level

In recent contributions we showed that interplay between fluorescence experiments (i.e. z-scan Fluorescence Correlation Spectroscopy (FCS) and solvent relaxation technique) and MD simulations allow controlling the membrane state on an atomistic level.1, 2, 3

In particular we focused on the role of ions4, 5 and oxidized phospholipids6, 7 on the biophysical membrane parameters like, lipid mobility, packing density and degree of hydration. Both directions are presently supported by the grants "Specific ion effects for proteins in solutions and related biologically relevant systems" and "Molecular level physiology and pathology of oxidized phospholipids (Eurocores)" from the Czech granting agencies, respectively, and both grants will end in 2012.

Especially, connected with the latter topic, we would like to stress its pharmacological background. Oxidized phospholipids have been shown to increase significantly in apoptosis, as well as in inflammation. Besides, they are involved in several pathological conditions, such as atherosclerosis, cancer, inflammation, Alzheimer's and Parkinson's disease and type 2 diabetes, with the detailed mechanisms remaining to be established.

We obtained a comprehensive picture how simple alkalications and halide anions, as well as selected oxidized control the physical state of lipid bilayers exclusively formed by simple phosphatidylcholines.

We plan to increase the complexity of the model membrane by adding physiological important membrane components like phosphatidylserines, phosphatidylethanoleamines, spinghomyelin, cholesterol, gangliosides, phosphatidylinositols, and ceramides. The listed lipids imply that we will not only characterize the impact on those above mentioned biophysical parameters, but that we will also address the influence of ions and oxidized phospholipids on phase separation phenomena, including the characterization of cholesterol-enriched nanodomains.

We are aware that in the latter very attractive research field the developments of suitable MD approaches lag behind the recent advances in (single molecule) fluorescence microscopy and spectroscopy. However, our experience from our collaborative work on those "simple" phosphatidylcholine bilayers teaches us that the combination of those approaches may yield an understanding of membrane biophysics on an atomistic level.

On top of that, the effect of oxidized phospholipids on model membranes will be paralleled by electrochemical investigation of membrane permeability on the single molecular or ion level, another key parameter in membrane biophysics.


References:

  1. Jurkiewicz, P.; Cwiklik, L.; Jungwirth, P.; Hof, M. Lipid hydration and mobility: An interplay between solvent relaxation experiments and molecular dynamic simulations. Biochimie, doi:10.1016/j.biochi.2011.06.027
  2. Olżyńska, A.; Zan, A.; Jurkiewicz, P.; Sýkora, J.; Grobner, G.; Langner, M. and M. Hof. Molecular interpretation of fluorescence solvent relaxation of Patman and H-2 NMR experiments in phosphatidylcholine bilayers. 2007, Chemistry and Physics of Lipids, 147(2): p. 69-77.
  3. Barucha-Kraszewska, J.; Kraszewski, S.; Jurkiewicz, P.; Ramseyer, Ch.; Hof, M. Numerical studies of the membrane fluorescent dyes dynamics in ground and excited states. 2010, Biochimica Et Biophysica Acta-Biomembranes 1798, 1724-1734.
  4. Vácha, R.; Siu, S.W.I., Petrov, M.; Böckmann, R.A.; Barucha-Kraszewska, J.; Jurkiewicz, P.; Hof, M.; Berkowitz, M.L.; and P. Jungwirth. Effects of Alkali Cations and Halide Anions on the DOPC Lipid Membrane. 2009, Journal of Physical Chemistry A, 113(26): p. 7235-7243.
  5. Vácha, R.; Jurkiewicz, P.; Petrov, M.; Berkowitz, M. L.; Böckmann, R. A.; Barucha-Kraszewska, J.; Hof, M.; Jungwirth, P. Mechanism of Interaction of Monovalent Ions with Phosphatidylcholine Lipid Membranes. 2010, Journal of Physical Chemistry B 114, 9504-9509.
  6. Beranová, L.; Cwiklik, L.; Jurkiewicz, P.; Hof, M.; Jungwirth, P. Oxidation Changes Physical Properties of Phospholipid Bilayers: Fluorescence Spectroscopy and Molecular Simulations. 2010, Langmuir 26, 61406144.
  7. Volinský, R.; Cwiklik, L. Jurkiewicz, P.; Hof, M.; Jungwirth, P.; Kinnunen, P.K.J. Oxidised Phosphatidylcholones reconstitute phospholipid "scramblase" activity in liposomes. Biophysical Journal, in press.
>> go to the top >>


Project 2:

Advanced Fluorescence Microscopy in Cell Biology

Our group has developed novel single molecule fluorescence techniques (i.e.: z-scan Fluorescence Correlation Spectroscopy - FCS1, 2; Fluorescence Lifetime Correlation Spectroscopy - FLCS3 and Dynamic Saturation Optical Microscopy - DSOM4), and implemented several cutting edge fluorescence techniques (i.e.: Fluorescence Lifetime Imaging - FLIM5; Raster Image Correlation Spectroscopy - RICS6; Photoactivation Localization Microscopy - PALM) in our lab.

Supported by the research centre "Advanced Fluorescence Microscopy in Biology" we attempted to apply these techniques in actual problems in Molecular Biology. As an example might serve the collaboration with David Staněk (IMG ASCR) focusing on the mechanism of the spliceosome assembly in the cell nucleus.5, 7

While we intend to continue our collaborations with several biological institutes within the ASCR (i.e.: IMG5, 6; IPG8, 9; IMB10; IEM6 and very recently also IEB) the arrival of Marek Cebecauer, a biologist with a long-term experience in cellular membrane studies, opened the possibility to develop our own research program in membrane biology employing those unique fluorescence techniques.

Specifically, three main topics will be studied:

  • The effect of transmembrane domains of integral proteins on dynamic organization of the plasma membrane T lymphocytes11;
  • Interaction of cholesterol-dependent cytolysin, perforin, with the plasma membrane, molecular organization therein and its impact on membrane properties;
  • Dynamics of membrane localization of regulatory kinase Csk and its impact on proximal T cell signaling.

All three topics characterize the membrane organization at the molecular level, which significantly influences the output of biological reactions in living organisms.


References:

  1. Benda, A.; Beneš, M.; Mareček, V.; Lhotský, A.; Hermens, W.T. and M. Hof. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. 2003, Langmuir, 19(10): p. 4120-4126.
  2. Macháň, R.; Hof, M. Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy. 2010, Biochimica Et Biophysica Acta-Biomembranes 1798, 1377-1391.
  3. Humpolíčková, J.; Benda, A.; Sýkora, J.; Macháň, R.; Kral, T.; Gasinska, B.; Enderlein, J. and M. Hof. Equilibrium dynamics of spermine-induced plasmid DNA condensation revealed by fluorescence lifetime correlation spectroscopy. 2008, Biophysical Journal, 94(3): p. L17-L19.
  4. Humpolíčková, J.; Benda, A.; Macháň, R.; Enderlein, J.; Hof, M. Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement. 2010, Physical Chemistry Chemical Physics 12, 12457-12465.
  5. Huranová, M.; Jablonski, J.A.; Benda, A.; Hof, M.; Staněk, D. and M. Caputi. In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer. 2009, Rna-a Publication of the Rna Society 15(11):2063-2071.
  6. Norris, S.C.P.; Humpolíčková, J.; Amler, E.; Huranová, M.; Buzgo, M.; Macháň, R.; Hof, M. Raster image correlation spectroscopy as a novel toll to study interactions of macromolecules with nanofiber scaffolds. Acta Biomaterialia, in press.
  7. Huranová, M.; Ivani, I.; Benda, A.; Poser, I.; Brody, Y.; Hof, M.; Shav-Tal, Y.; Neugebauer, K.; Staněk D. The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. 2010, Journal of Cell Biology 191, 75-86.
  8. Sýkora, J.; Bouřová, L.; Hof, M. and P. Svoboda. The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: Correlation with studies of DPH and Laurdan fluorescence. Biochimica Et Biophysica Acta-Biomembranes, 2009. 1788(2): p. 324-332.
  9. Brejchová, J.; Sýkora, J.; Roubalová, L.; Ostašov, P.; Vošahlíková, M.; Hof, M.; Svoboda, P. Fluorescence spectroscopy studies of HEK293 cells expressing DOR- Gi1α fusion protein; the effect of cholesterol depletion. Biochimica Et Biophysica Acta-Biomembranes, revised version submitted.
  10. Hovorka, O.; Šubr, V.; Větvička, D.; Kovář, L.; Strohalm, J.; Strohalm, M.; Benda, A.; Hof, M.; Ulbrich, K.; Říhová, B. Spectral analysis of doxorubicin accumulation and the indirect quantification of its DNA intercalation. 2010, European Journal of Pharmaceutics and Biopharmaceutics, 76, 514-524.
  11. Owen, D.M.; Oddos, S.; Kumar, S.; Davis, D.M.; Neil, M.A.; French, P.M.; Dustin, M.L.; Magee, A.I.; Cebecauer, M. High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells. 2010, Molecular Membrane Biology, 27(4-6):178-89.
>> go to the top >>


Project 3:

Elucidating the Dynamic/Hydration-Function Relationships in Hydrolytic Enzymes

Enzymes are widely used for the synthesis of pharmaceuticals, agrochemicals and food additives because of their ability to catalyze enantioselective transformations. Understanding the molecular basis of enzyme-substrate interactions that contribute to enantioselectivity is important for constructing selective enzymes by protein engineering.

An emerging group of enzymes that is explored for enatioselectivity are dehalogenases. Haloalkane dehalogenases can convert a broad range of halogenated aliphatic substrates to their corresponding alcohols, and because of the simplicity of the reaction represent a good model system to study structural basis of reactivity and enantioselectivity.

In our joint efforts with the group of Jiří Damborský (MU Brno) we were able to selectively label the active site of this enzyme class. We showed that fluorescent solvent relaxation technique in synergy with MD simulations allows the characterization of hydration and dynamics at the entry to the active site pocket.1

Our very recent experiments strongly indicate that these two parameters are linked to the enantioslectivity of certain designed dehalogenase enzymes. As this finding is important for the overall concept of protein design, we will expand this unique approach, which is based on the combination of the solvent relaxation technique, MD simulations, as well as protein design and characterization, to other well selected representative of this protein class with the aim to make more general conclusions which will be essential for the entire concept of de novo protein design.

On top of that, these experiments will be expanded to organic solvents as well as ions and their role in that dynamic/hydration-function relationship.


References:

  1. Jesenská, A., J. Sýkora, A. Olżyńska, J. Brezovský, Z. Zdráhal, J. Damborský, and M. Hof. Nanosecond Time-Dependent Stokes Shift at the Tunnel Mouth of Haloalkane Dehalogenases. Journal of the American Chemical Society, 2009. 131(2): p. 494-501.
>> go to the top >>