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


Project 1:

Understanding Membrane Biophysics on an Atomistic Level

Presently three main research directions in membrane biophysics might be defined:

i) Investigation of the role of the lipid nanodomains in biology.

Nanodomains are prepared in model phospholipid bilayers under controlled conditions. The combination of Foerster energy transfer and Monte-Carlo simulations enable to monitor their presence and dimensions. The effect of these nanodomains on the interaction and the behavior of various molecules (e.g. gangliosides, beta-amyloids, oxidized phospholipids, toxins) and vice versa is then followed mainly by the approaches based on the fluorescence microscopy (z-scan, dual-foci, and cross-correlation, FCS, Number & brightness analysis, fluorescence anti-bunching ).

Some selected recent publications:

  1. Štefl, M.; Šachl, R.; Humpolícková, J.; Cebecauer, M.; Machán, R.; Kolárová, M.; Johansson, L.B.-Ĺ.; Hof, M.Dynamics and Size of Cross-Linking-Induced Lipid Nanodomains in Model Membranes. 2012, Biophysical Journal 102, 9, 2104-2113. DOI: 10.1016/j.bpj.2012.03.054.
  2. Šachl, R.; Humpolícková; J.; Štefl, M.; Johansson, L.B.-A; Hof, M. Limitations of Electronic Energy Transfer in Lipid Nanodomain Size Estimation. 2011, Biophys. J., 101, L60-L62.

    3. ii) Changes in biomembrane hydration and packing upon the interactions with ions and small molecules.

    Model phospholipid bilayers mimicking the biomembranes are exposed to various ions, drugs or peptides. The changes in the hydration and packing are monitored by Time dependent fluorescence shift, which is a bulk technique combining time-resolved fluorescence with the stationary one. Gained data are then compared with the results obtained by molecular dynamics simulations providing a complex view of the bilayer arrangements.

    Some selected recent publications:

    1. Först, G.; Cwiklik, L.; Jurkiewicz, P.; Schubert, R.; Hof, M. Interactions of beta-blockers with model lipid membranes: Molecular view of the interaction of acebutolol, oxprenolol, and propranolol with phosphatidylcholine vesicles by time-dependent fluorescence shift and molecular dynamics simulations. . 2014, Eur. J. Pharm. Biopharm. DOI: 10.1016/j.ejpb.2014.03.013.
    2. Machán, R.; Jurkiewicz, P.; Olzynska, A.; Olšinová, M.; Cebecauer, M.; Marquette, A.; Bechinger, B.; Hof, M.; Peripheral and Integral Membrane Binding of Peptides Characterized by Time-Dependent Fluorescence Shifts: Focus on Antimicrobial Peptide LAH4. 2014, Langmuir, 30(21), 6171-6179.
    3. Vazdar, M.; Wernersson, E.; Khabiri, M.; Cwiklik, L.; Jurkiewicz, P.; Hof, M.; Mann, E.; Kolusheva, S.; Jelinek, R.; Jungwirth, P. Aggregation of Oligoarginines at Phospholipid Membranes: Molecular Dynamics Simulations, Time-Dependent Fluorescence Shift, and Biomimetic Colorimetric Assays. 2013, J. Phys. Chem. B 117, 39, 11530-11540.
      DOI: 10.1021/jp405451e
    4. Pokorná, Š.; Jurkiewicz, P.; Cwiklik, L.; Vazdar, M. and Hof, M. Interactions of monovalent salts with cationic lipid bilayers. 2013, Faraday Discuss. 160, 341-358.
      DOI: 10.1039/C2FD20098H
    5. Beranová, L.; Humpolíčková, J.; Sýkora, J.; Benda, A.; Cwiklik, L.; Jurkiewicz, P.; Gröbner, G.; Hof, M. Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations. 2012, Phys. Chem. Chem. Phys. 14, 42, 14516-14522.
      DOI: 10.1039/C2CP41275F
    6. Jurkiewicz, P.; Cwiklik, L.; Vojtíšková, A.; Jungwirth, P.; Hof, M. Structure, Dynamics, and Hydration of POPC/POPS Bilayers Suspended in NaCl, KCl, and CsCl Solutions. 2012, BBA - Biomembranes 1818, 3, 609-616.
      DOI: 10.1016/j.bbamem.2011.11.033
    7. 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.

      8. iii) Changes in biomembrane biophysics induced by lipid and sterol oxidation.

      Model phospholipid bilayers mimicking the biomembranes are enriched with oxidized phospholipids or oxy-sterols. The changes in the membrane biophysics are determined by the time dependent fluorescence shift technique as well as calibration free FCS techniques. Gained data are then compared with the results obtained by molecular dynamics simulations providing a complex view of the bilayer arrangements.

      Some selected recent publications:

      1. Štefl, M.; Šachl, R.; Olżyńska, A.; Amaro, M.; Savchenko, D.; Deyneka, A.; Hermetter, A.; Cwiklik, L.; Humpolíčková, J.; Hof, M. Comprehensive portrait of cholesterol containing oxidized membrane. 2014, BBA - Biomembranes, 1838, 7, 1769-1776.
        DOI: 10.1016/j.bbamem.2014.02.006
      2. Jurkiewicz, P.; Olżyńska, A.; Cwiklik, L.; Conte, E.; Jungwirth, P.; Megli, F.M.; Hof M. Biophysics of lipid bilayers containing oxidatively modified phospholipids: Insights from fluorescence and EPR experiments and from MD simulations. 2012, BBA - Biomembranes 1818, 10, 2388-2402.
        DOI: 10.1016/j.bbamem.2012.05.020
      3. Vazdar, M.; Jurkiewicz, P.; Hof, M.; Jungwirth, P.; Cwiklik, L. Behavior of 4-Hydroxynonenal in Phospholipid Membranes. 2012, Journal of Physical Chemistry B 116, 22, 6411-6415.
        DOI: 10.1021/jp3044219
      4. Volinsky, R.; Cwiklik, L.; Jurkiewicz, P.; Hof, M.; Jungwirth, P.; Kinnunen, P.K.J. Oxidized Phosphatidylcholines Facilitate Phospholipid Flip-Flop in Liposomes. 2011, Biophysical Journal, 101, 6, 1376-1384.
        DOI: 10.1016/j.bpj.2011.07.051
      5. 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, 6140-6144.
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      Project 2:

      Advanced Fluorescence Microscopy: Development of Techniques and Applications

      Our group has developed or contributed to development of novel single molecule fluorescence techniques (i.e.: z-scan Fluorescence Correlation Spectroscopy (z-FCS); Fluorescence Lifetime Correlation Spectroscopy (FLCS), Fluorescence Spectral Correlation Spectroscopy (FSCS), and Dynamic Saturation Optical Microscopy (DSOM)). Moreover, the group implemented cutting edge fluorescence techniques (e.g. raster image correlation spectroscopy (RICS), photoactivation localization microscopy (PALM), cross correlation Number & Brightness analysis) and further improved such already existing techniques (fluorescence lifetime imaging (FLIM/FRET) for quantitative size determination of nanodomains, fluorescence antibunching for membrane-associated aggregation phenomena, and dual-focus FCS).

      Previously 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 Stanek (IMG ASCR) focusing on the mechanism of the spliceosome assembly in the cell nucleus.

      Some selected recent publications:

      1. Benda, A.; Kapusta, Peter; Hof, Martin; Gaus, K. Fluorescence spectral correlation spectroscopy (FSCS) for probes with highly overlapping emission spectra. 2014, Opt. Express, 22, 3, 2973-2988.
        DOI: 10.1364/OE.22.002973
      2. Štefl, M.; Benda, A.; Gregor, I. and Hof, M. The fast polarization modulation based dual-focus fluorescence correlation spectroscopy. 2014, Opt. Express, 22, 1, 885-899.
        DOI: 10.1364/OE.22.000885
      3. Hnilicová, J.; Hozeifi, S.; Stejskalová, E.; Dušková, E.; Poser, I.; Humpolíčková, J.; Hof, M.; Staněk, D. The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing. 2013, Mol Biol Cell. 24, 22, 3557-3568.
        DOI: 10.1091/mbc.E13-06-0303
      4. Ostašov, P.; Sýkora, J.; Brejchová, J.; Olżyńska, A.; Hof, M.; Svoboda, P. FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: Plasma membrane change induced by cholesterol depletion 2013, Chemistry and Physics of Lipids, 167-168, 62-69.
        DOI: 10.1016/j.chemphyslip.2013.02.006
      5. Strachotová, D.; Holoubek, A.; Kučerová, H.; Benda, A.; Humpolíčková, J.; Váchová, L.; Palková, Z. Ato protein interactions in yeast plasma membrane revealed by fluorescence lifetime imaging (FLIM). 2012, BBA - Biomembranes 1818, 9, 2126-2134.
        DOI: 10.1016/j.bbamem.2012.05.005
      6. Dohnalová, K.; Fučíková, A.; Umesh, Ch. P.; Humpolíčková, J.; Paulusse, J. M. J.; Valenta, J.; Zuilhof, H.; Hof, M.; Gregorkiewicz, T. Microscopic Origin of the Fast Blue-Green Luminescence of Chemically Synthesized Non-oxidized Silicon Quantum Dots. 2012, Small 8, 20, 3185-3191.
        DOI: 10.1002/smll.201200477
      7. 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.
      8. 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.
      9. 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.
      10. 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.
      11. Pembouong, G.; Morellet, N.; Kral, T.; Hof, M.; Scherman, D.; Bureau, M. F.; Mignet, N. A comprehensive study in triblock copolymer membrane interaction. 2011, Journal of Controlled Release 151, 1, 57-64.
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      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.

      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, connected with that topic we are developing new probes and methods for probing dynamic and hydration in enzymes.

      Additionally in cooperation with professor Antonín Vlcek, we try to elucidate the relationship between the dynamic/hydration of the particular protein region and the ability of the proteins to transfer electrons on long range distances. The coupling between the electron-transfer and structural relaxation dynamics is monitored by means of time-dependent fluorescence shifts and compared with the time resolved infrared spectroscopy in azurins and cytochromes using both the organic and complex anorganic chromophores. The study shall elucidate the mechanisms that enable proteins to transfer electrons on nanometer scales and help to develop efficient fotocatalytic systems.


      Some selected recent publications:

      1. Sýkora, J.; Brezovský, J.; Koudeláková, T.; Lahoda, M.; Fořtová, A.; Chernovets, T.; Chaloupková, R.; Štěpánková, V.; Prokop, Z.; Smatanová, I.K.; Hof, M.; Damborský, J. Dynamics and hydration explain failed functional transformation in dehalogenase design. 2014, Nat. Chem. Biol.
        DOI: 10.1038/nchembio.1502
      2. Amaro, M.; Brezovský, J.; Kováčová, S.; Maier, L.; Chaloupková, R.; Sýkora, J.; Paruch, K.; Damborský, J. and Hof, M. Are Time-Dependent Fluorescence Shifts at the Tunnel Mouth of Haloalkane Dehalogenase Enzymes Dependent on the Choice of the Chromophore? 2013, J. Phys. Chem. B, 117 (26), 7898-7906.
        DOI: 10.1021/jp403708c
      3. Štěpánková, V.; Khabiri, M.; Březovský, J.; Pavelka, A.; Sýkora, J.; Amaro, M.; Minofar, B.; Prokop, Z.; Hof, M.; Ettrich, R.; Chaloupková, R.; Damborský, J. Expansion of Access Tunnels and Active-Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents. 2013, Chembiochem. 14, 7, 890-897.
        DOI: 10.1002/cbic.201200733
      4. 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.
      5. Blanco-Rodríguez, A.M.; Di Billio, A.J.; Shih, C.; Museth, A.K.; Clark, I.P.; Towrie, M.; Cannizzo, A.; Sudhamsu, J.; Crane, B.R.; Sýkora, J.; Winkler, J.R.; Gray, H.B.; Záliš, S.; Vlček Jr.A. Phototriggering Electron Flow through ReI-modified Pseudomonas aeruginosa Azurins. 2011, Chemistry - A European Journal 17, 19, 5350-5361.
      6. Blanco-Rodriguez, A.M., M. Busby, K. Ronayne, M. Towrie, C. Gradinaru, J. Sudhamsu, J. Sýkora, M. Hof, S. Záliš, A.J. Di Bilio, B.R. Crane, H.B. Gray, and A. Vlček. Relaxation Dynamics of Pseudomonas aeruginosa Re-I(CO)(3)(alpha-diimine)(HisX)(+) (X=83, 107, 109, 124, 126)Cu-II Azurins. 2009, Journal of the American Chemical Society 131(33):11788-11800.
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