Charles University in Prague Faculty of Mathematics and Physics DOCTORAL THESIS. Lenka Beranová - PDF

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Charles University in Prague Faculty of Mathematics and Physics DOCTORAL THESIS Lenka Beranová Advanced fluorescence techniques applied on biomolecules (lipid membranes and DNA) J. Heyrovský Institute

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Charles University in Prague Faculty of Mathematics and Physics DOCTORAL THESIS Lenka Beranová Advanced fluorescence techniques applied on biomolecules (lipid membranes and DNA) J. Heyrovský Institute of Physical Chemistry of the ASCR, v. v. i. Supervisor of the doctoral thesis: Prof. Martin Hof, DSc. Study programme: Physics Specialization: Biophysics, Chemical and Macromolecular Physics Prague 2013 I would like to express my gratitude to many people. First of all, to my supervisor Martin Hof. I have many reasons for it, but what I will mention here is his friendly approach to me, his optimism and moral support. Many thanks to all my colleagues from J. Heyrovský Institute (Piotr, Honza S., Jana, Agnieszka, Radek Š., Radek M., Teresa, Šárka, Aleš, Martin Š., Tomáš, Marek, Marie, Alžběta, Marianna) who were all the time prepared to help me. Thank you for everything I could learn from you, for your enthusiasm I could experience, for your encouragement and for all the fun we had together. I owe my gratitude to Gerhard Gröbner from Umeå who invited me to his laboratory. Thank you for the fresh air which came into my research efforts during the stay in Sweden, for the new ideas and for the fact you provided me there with everything needed including a bicycle and many social contacts. I am deeply indebted to my family who unconditionally supported me all the time. Many thanks also to my friends to be there for me, especially to Eva S., Eva R. and Eva Q. who, together with my family, provided me with encouragement. Thanks to Honza who was with me during the finishing. Finally, many thanks to God for giving me unexpected gifts. I declare that I carried out this doctoral thesis independently, and only with the cited sources, literature and other professional sources. I understand that my work relates to the rights and obligations under the Act No. 121/2000 Coll., the Copyright Act, as amended, in particular the fact that the Charles University in Prague has the right to conclude a license agreement on the use of this work as a school work pursuant to Section 60 paragraph 1 of the Copyright Act. In Prague on 28 th June 2013 Lenka Beranová Název práce: Pokročilé fluorescenční metody aplikované ve výzkumu biomolekul (lipidových membrán a DNA) Autorka: Lenka Beranová Katedra / Ústav: Ústav fyzikální chemie J. Heyrovského AV ČR, v. v. i. Vedoucí disertační práce: prof. Martin Hof, DSc., Ústav fyzikální chemie J. Heyrovského AV ČR, v. v. i. Abstrakt: Tato práce popisuje dvě pokročilé fluorescenční metody: metodu zkoumající časový vývoj Stokesova posunu fluorescenčního spektra (TDFS) a fluorescenční korelační spektroskopii (FCS) včetně jejích speciálních obměn - časově rozlišené FCS, Z-scan FCS a dvojohniskové FCS. Metody byly aplikovány ve výzkumu DNA a lipidových membrán. Přispěli jsme k objasnění mechanismu kondenzace molekul DNA menších, než je rozlišovací schopnost konfokálního mikroskopu. Kondenzace polykationem sperminem je zřejmě proces diskrétní ( all or non ), při použití kladně nabitého surfaktantu kondenzace probíhá naopak postupně. Dále jsme zkoumali biofyzikální vlastnosti fosfolipidové membrány ovlivněné přítomností oxidovaných lipidů se zkráceným sn-2 řetězcem. Jejich vlivem v membráně vzrostla pohyblivost a hydratace v oblasti lipidových hlaviček. Tento efekt je ve shodě s molekulovými simulacemi, které ukázaly reorientaci krátkých sn-2 řetězců oxidovaných lipidů. Přítomnost oxidovaných molekul může také ovlivňovat difuzi lipidů v membráně, pozorovali jsme mírně zvýšený difuzní koeficient. Zkoumali jsme také vliv těžké vody na fosfolipidovou membránu. Přítomnost D 2 O způsobuje snížení pohyblivost a hydratace oblasti lipidových hlaviček, což je v souladu se simulacemi, které ukazují zvýšení dob života vodíkových vazeb mezi molekulami vody a molekulami lipidů. Klíčová slova: kondenzace DNA, oxidované lipidy, těžká voda, fluorescenční korelační spektroskopie, časový vývoj posunu fluorescenčního spektra Title: Advanced fluorescence techniques applied on biomolecules (lipid membranes and DNA) Author: Lenka Beranová Department / Institute: J. Heyrovský Institute of Physical Chemistry of the ASCR, v. v. i. Supervisor: prof. Martin Hof, DSc., J. Heyrovský Institute of Physical Chemistry of the ASCR, v. v. i. Abstract: The thesis describes time dependent fluorescence shift method and fluorescence correlation spectroscopy method (FCS) with its extensions FLCS, Z-scan FCS and dualfocus FCS applied on specific problems in DNA and lipid research. Compaction mechanism of a DNA molecule smaller than a resolution of a confocal microscope was elucidated. The process was revealed to be all or non for a polycation spermine as a condenser in contrast with the gradual compaction caused by a cationic surfactant. Biophysical properties of a phospholipid bilayer influenced by presence of oxidized phospholipids with truncated sn-2 chain were explored. The dynamics of hydrated functional groups in the headgroup region was proved to get faster while the hydration of the headgroup region increased. These effects are in relation with the reorientation of the short sn-2 chains observed in molecular dynamics simulations. Presence of oxidized species may also influence the lateral diffusion of the lipids a slight increase of the diffusion coefficient was observed. Decrease of hydration and mobility in the headgroup region was found as an influence of heavy water on the phospholipid membrane. These finding are in line with molecular dynamics simulations which show longer lifetimes of hydrogen bonds between water and lipid molecules in presence of heavy water. Keywords: DNA compaction, oxidized lipids, heavy water, fluorescence correlation spectroscopy, time dependent fluorescence shift Contents 1. INTRODUCTION ADVANCED FLUORESCENCE METHODS USED IN DNA AND LIPID MEMBRANES STUDIES Fluorescence correlation spectroscopy and its advanced extensions Principle of fluorescence correlation spectroscopy Fluorescence lifetime correlation spectroscopy FCS in lipid membranes Z-scan FCS Dual-focus FCS Hydration and mobility studied by time dependent fluorescence shift method (TDFS) Fluorescence emission spectrum and the influence of the solvent Time dependent fluorescence shift method TDFS in membranes COMPACTION MECHANISM OF A LINEAR DNA MOLECULE INDUCED BY SPERMINE AND CTAB DNA compaction process FCS and FLCS approach to DNA compaction Compaction mechanism of a 48.5 kbp long linear DNA molecule induced by spermine or CTAB INFLUENCE OF OXIDIZED LIPIDS ON PHYSICAL PROPERTIES OF PHOSPHOLIPID BILAYER Introduction to oxidized lipids TDFS measurements of hydration and mobility Z-scan FCS exploring lateral diffusion Molecular dynamics simulations Further fluorescence studies of oxidized lipids...30 5. EFFECT OF HEAVY WATER ON PHOSPHOLIPID MEMBRANES Introduction the the effect of heavy water on biomolecules Specific biophysical properties of a phospholipid bilayer influenced by heavy water Differential scanning calorimetry: shift in phase transition temperature Time-dependent fluorescence shift: mobility and hydration of the headgroup region Steady state and time-resolved fluorescence anisotropy: lipid order parameter Two-focus fluorescence correlation spectroscopy: lateral lipid diffusion coefficient Summary of the effect of heavy water on phospholipid membrane CONCLUSION BIBLIOGRAPHY LIST OF TABLES LIST OF ABBREVIATIONS APPENDICES...56 1. Introduction 1 Introduction Since the seminal contributions by Gregorio Weber in the second part of the last century, it became clear that fluorescence is a useful tool in the studies of biomolecules. The vast progress within the last decade in instrumentation and development of new concepts, however, promoted fluorescence to be the main tool when it comes to studies of dynamics and organisation of biomolecules, biomolecular assembles, and living cells. There is a relatively large number of emerging new techniques having single molecule sensitivity, nanometer resolution, or resolving picoseconds processes and some of them were developed or implemented in our group. In this work, some of those methods are described and applications in DNA and lipid bilayer research are presented. Specifically, fluorescence correlation spectroscopy (FCS) is an advanced application of fluorescence microscopy based on analysis of fluctuations of fluorescence light coming from the detection volume of a confocal microscope. It allows determination of diffusion properties of fluorescently labeled molecules of nanomolar concentrations in solutions and even in living cells. Moreover, fluorescence lifetime correlation spectroscopy enables distinguishing between diffusing species with different lifetimes. Due to this specific benefit it was possible to elucidate the compaction mechanism of a DNA molecule smaller then a resolution of a confocal microscope (see chapter 3 and appendix 1). Fluorescence correlation spectroscopy is a suitable tool also for measuring lateral diffusion of lipids in a bilayer. Its advanced extensions, which does not need any external calibration, Z-scan FCS and dual-focus FCS were employed to measure lateral diffusion in membranes influenced by presence of oxidized lipids (see chapter 4 and appendix 4) and in the bilayer affected by heavy water (section 5 and appendix 5). Time dependent fluorescence shift method explores the time evolution of the emission spectrum of a fluorescent dye. In membranes, this process occurs on nanosecond time scale and reflects the rearrangement of the hydrated functional 1 1. Introduction groups of phospholipid molecules. The total spectral shift mirrors the polarity of the chromophore s environment which is closely related to the level of hydration of the corresponding part of the bilayer. The changes in hydration and local mobility in the headgroup region of membranes containing oxidized lipids were explored (section 4 and appendix 4). The effect of heavy water on the biophysical properties of a bilayer was also investigated using this method (see section 5 and appendix 5). 2 2. Advanced fluorescence methods used in DNA and lipid membranes studies 2 Advanced fluorescence methods used in DNA and lipid membranes studies 2.1 Fluorescence correlation spectroscopy and its advanced extensions Principle of fluorescence correlation spectroscopy A paper containing comprehensive description of fluorescence correlation spectroscopy method in Czech language is to be found in appendix 2. Fluorescence correlation spectroscopy [1, 2] is an advanced microscopic technique which is based on analysis of fluctuations of fluorescence light coming from the detection volume of a confocal microscope. The fluctuations arise mainly from diffusion of fluorescently labeled molecules across the focal volume (and this is the property we were focused on) but can be also caused by other physicochemical processes and therefore investigate them (flow, chemical reactions, transition to a nonfluorescent state). The signal-to-noise ratio in FCS is optimal when there is approximately one or very few individual fluorescent molecules in the focal volume of the microscope. Therefore the concentration of the fluorescent molecules is usually very low and FCS is considered as single molecule technique. The standard experimental setting is depicted on Figure 1. It involves confocal microscope with high numerical aperture objective and sensitive single-photon detector. (ACF): The fluorescent signal is correlated by means of an autocorrelation function G ( τ ) ( t) δi( t + τ ) I( t) 2 δi = (1) where I(t) is the intensity of fluorescence at time t, τ is the lag-time, stands for the mean value over the time of the measurement and (2) ( t) = I( t) I( t) δ I. (3) A more detailed explanation of the autocorrelation is given on Fig 2. 3 2. Advanced fluorescence methods used in DNA and lipid membranes studies Fig. 1: Scheme of confocal microscope The autocorrelation function has to be fitted to an a priori known mathematical model that describes the processes leading to the fluctuations, for freely diffusing uniform particles it is: G ( τ ) 1 1 = PN 1+ ( τ / τ ) 1 1 ( 1+ ( τ / τ )( r z ) ) 2 2 D / D 0 where PN ( particle number ) stands for average number of diffusing species within the focal volume, τ D is the mean residence time of a particle within the focal volume, r 0 and z 0 are half axes of the focal volume. From the residence time τ D one can calculate the diffusion coefficient D of the molecule (a parameter which is not dependent on the particular experimental setting) if the size and shape of the focal volume are known. As these parameters are usually unknown, external calibration using a solution of particles with a known diffusion coefficient is needed for getting the diffusion coefficient. 0 (4) 4 2. Advanced fluorescence methods used in DNA and lipid membranes studies Fig. 2: Explanation of the autocorrelation function: A measured fluctuations of fluorescence, B calculated autocorrelation function. For short lag-times (τ 1 ) the autocorrelation is high, for longer intervals (τ 2 ) it is going down because the intensity values at the beginning and at the end of the interval are not related. τ D is average residence time of a particle in the focal volume. For this particular example: PN = 3, τ D = 54 ms, z 0 /r 0 = 7. When the diffusion is not the only process which affects the autocorrelation function, e.g. the molecule undergoes transition to a non-fluorescent triplet state and back during the diffusion through the focal volume, the autocorrelation function has to be modified and further parameters fitted. Crosscorrelation is a correlation between two different signals. It is used for overcoming afterpulsing of the detectors, a drawback which detectors used for FCS measurements often suffer from. This means that genuine output pulses are followed 5 2. Advanced fluorescence methods used in DNA and lipid membranes studies by an afterpulse. This secondary phenomenon is correlated to an initial output pulse so the autocorrelation function is seriously influenced by this artefact, especially in the area of shorter delay times. This drawback is often overcome by splitting the emission light on two different detectors and crosscorrelating their outputs. Other more sophisticated applications of FCS use autocorrelations and crosscorrelations of two fluorescent signals coming from two different dyes with different emission wavelengths (dual colour FCS [3, 4]) or with different lifetime (FLCS - fluorescence lifetime correlation spectroscopy) [5, 6] Fluorescence lifetime correlation spectroscopy A review of fluorescence lifetime correlation spectroscopy is to be found in appendix 3. Fluorescence lifetime correlation spectroscopy [5, 6] enables to correlate signals coming from two dyes with different lifetime (alternatively it can be the same dye located in different environments which results in having different lifetime). Additional equipment is required in comparison with standard FCS: sub-nanosecond pulsed excitation instead of continuous wave illumination is needed. The second requirement is the ability to measure the fluorescence photon arrival time in the special time-tagged time-resolved mode. This means to measure on two different scales: 1) relative to the last excitation pulse with picosecond resolution this so-called microtime contains information about the fluorescence decay, 2) relative to the start of the experiment with nanosecond precision this so-called macrotime is measured on continuous time axes and contains information related e.g. to diffusion motion just like in standard FCS. Consequently, the measured data consist of a list of all detected photons with two different arrival time values microtime and macrotime see Fig. 3. 6 2. Advanced fluorescence methods used in DNA and lipid membranes studies laser pulses photon 1 photon 2 microtime of photon 1 microtime of photon 2 [ps] macrotime of photon 1 macrotime of photon 2 [ns] Fig. 3: Demonstration of measuring on two different time-scales: microtime with respect to last laser pulse, macrotime with respect to the beginning of the measurement. Consider a sample with two fluorescent components (in general it is possible to do it for more than two), which have different fluorescence lifetimes. The fluorescence decay histograms ( decay patterns ) of both components are known (e. g. from measurements on pure samples). From the measurement of photon arrival times on microtime scale one can get the fluorescence decay of the mixture of both components (see Fig. 4). Fig. 4: Fluorescence decays of two fluorophores (1 and 2) and the decay of a mixture of these two components 7 2. Advanced fluorescence methods used in DNA and lipid membranes studies At every macroscopic arrival time t (macrotime) and in each channel j 1 (microtime) the fluorescence intensity I j (t) is a linear combination of patterns p j and 2 p j (these are normalized lifetime decays of the two fluorophores): j ( t) w ( t) p 1 w 2 ( t) p 2 I = +, (5) 1 j j where w 1 (t) and w 2 (t) are the contributions of the individual fluorophores to the total fluorescence signal. These individual contributions are needed for calculation of the autocorrelation function of individual species. Solving the equation (5) we get w k N k ( t) = f I ( t) j= 1 j j, (6) k where k is the number of the component, N is the number of channels and f j is a discrete filter function, which is constructed from the fluorescence decay histograms of the different fluorescence species and the time-averaged histogram of the compound signal. Explicitly, f j k is given by: f k j = ) T 1 ) 1 ) T [ M diag I j ( t) M ] M diag I j ( t) 1 t t kj, (7) f 1 filter functions f channels Fig. 5: Filter functions for two fluorophores (1 fluorophore with shorter lifetime, 2 fluorophore with longer lifetime) 8 2. Advanced fluorescence methods used in DNA and lipid membranes studies where the matrix elements are: M ˆ =. (8) k jk p j The filters corresponding to the decays depicted in Fig. 4 are plotted in Fig. 5. Due to the high filter function at the beginning of the decay, photons with small channel number are more probable to be emitted by fluorophore 1. On the other hand, it is more probable that a photon coming in later microtime (larger channel number) belongs to fluorophore 2. Using these filters, it is possible to obtain fluorescent intensity trace w(t) for all the components, which can be further autocorrelated or crosscorrelated. Autocorrelation of the k th component is calculated as: G k ( τ) w k ( t) w ( t + τ ) ( t) k t i= 1 j= 1 = = k 2 N N w t N N i= 1 j= 1 f k i f k i f f k j k j I I i ( t) I ( t + τ) i j ( t) I ( t) t j t t. (9) Using FLCS approach, several drawbacks of FCS can be overcome, e.g. the contribution of detector afterpulsing or light scattering can be filter out [6, 7]. Further interesting use of this method involves distinguishing between the molecules of the same dye surrounded by different microenvironment and thus having different lifetime. The processes leading to the fluorescence fluctuations can be analysed separately for these two different fractions. The change in lifetime can be caused by presence of light-absorbing surface [8], conformation change in a dye-protein complex [9], metal-fluorophore interaction [10] or different conformation of DNA at the place where the dye is intercalated [11]. Furthermore, it was proven that the separation is possible to realize even when the patterns of some components are inaccessible, specifically, one can filter out all the components which have a known pattern [12]. Suppose the system consists of two lifetime patterns p 1 and p 2, but the pattern p 2 is not experimentally accessible, i.e., we can me
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