Ph.D. Topics of the Institute of Biophysics

Structure and function of metal containing proteins

Group: Laboratory of Metalloprotein Biophysics
Supervisor: Csaba BAGYINKA

Purification and characterization of redox metalloenzymes from photosynthetic bacteria. Their investigation with different spectroscopic methods (CD, FTIR, UVVis, EPR, etc.). The ideal candidate is familiar with the cultivation of bacteria, knows the different protein purification methods and experienced in the different spectroscopic methods. If not he/she can learn it.

Investigation of the autocatalytic enzyme reaction in the hydrogenase enzyme

Group: Laboratory of Metalloprotein Biophysics
Supervisor: Csaba BAGYINKA

The main task of the project is the investigation of the autocatalytic and oscillatory enzyme reaction of the hydrogenase enzyme, and model construction describing the experimental data. It is a must to contribute in the purification, preparation of the enzyme. The ideal candidate is familiar with the cultivation of bacteria, knows the different protein purification methods. He/she is able to construct mathematical models, solve the differential equation systems, determine the parameters best describing the experiments. If not, he/she is able to learn it.

Purification of reaction centers from photosynthetic bacteria

Group: Laboratory of Metalloprotein Biophysics
Supervisor: Csaba BAGYINKA

The aim of the work is to purify and crystallize bacterial photosynthetic reaction centres from purple sulphur photosynthetic bacteria. The structure of this type of photosynthetic reaction centres is not known yet. The ideal candidate is familiar with microbiological methods, have used and familiar with the continuous flow photofermentor. He/she can work in sterile environment, knows the different separation and purification techniques. Able to learn the methods used for crystallization and apply for the crystallization of this type of reaction centres.

Purification and characterization of membrane-associated redox proteins

Group: Laboratory of Metalloprotein Biophysics
Supervisor: Alajos BÉRCZI

About 30% of proteins in cells are “associated” to membranes. Purification of these membrane-associated proteins always represents some challenge for scientists. In the frame of this project, redox proteins associated to or integrated in biological membrane structures are to be purified by different methods of centrifugation and chromatography. Biophysical and biochemical characterization (mostly by different spectroscopy) of the purified proteins is to be carried out both in detergent micelles and in a reconstituted form in proteo-liposomes. At present, the redox proteins under interest are b-type cytochromes of the cytochrome b561 protein family found originally in mouse and Arabidopsis.

Purification and characterization of cytochrome b561 proteins

Group: Laboratory of Metalloprotein Biophysics
Supervisor: Alajos BÉRCZI

The cytochrome b561 proteins (Cyts-b561) are members of a new protein family. These proteins are integral membrane proteins with 6 trans-membrane helices and two b-type hemes. More than one isoform of Cyts-b561 can be present in both plant and animal organisms. Until now, however, only one member of this protein family – namely the CGCytb in the chromaffin granule membranes in adrenal glands – has been characterized in some details. We are at present interested in the purification and characterization of two new members (namely the Cyb561d1 and Cyb561d2) of the Cyts-b561 by using recombinant proteins and different biophysical, biochemical, and molecular biology techniques.

Cell culture models to study drug permeability across barriers

Group: Laboratory of Molecular Neurobiology
Supervisor: Mária DELI

Two biological barriers are investigated with the help of primary cell cultures and immortalized cell lines. The blood-brain barrier, which controls the penetration of pharmacons from blood to the central nervous system and the nasal epithelium, which enables drug delivery from the nasal cavity to the systemic circulation. The in vitro bloodbrain barrier model reconstituted from brain endothelial cells, pericytes and glial cells, and the nasal epithelial cell model cultured on air/liquid interface are used to test the permeability and effects of drugs and excipients and estimate or predict the penetrability of drug candidates through barriers in humans. The models are suitable to optimize drug molecules or vehicles for drug delivery or targeting.

Investigation of blood-brain barrier permeability and transport in physiological and pathological conditions

Group: Laboratory of Molecular Neurobiology
Supervisor: Mária DELI

The blood-brain barrier have four general functions, nutrition of the brain, maintenance of homeostasis for neuronal functions, protection of the brain especially from toxins and bidirectional signaling between the nervous system and the periphery. The tight interendothelial barrier and different transport systems, including solute carriers, efflux pumps and receptor-mediated transport provide the basis to these functions. In pathological conditions, like ischemia, stroke, infections and tumors of the nervous systems, neurodegenerative disorders as Alzheimer’s and prion diseases blood-brain barrier functions can be damaged contributing to neuronal loss and disease progression. Our goal is (i) to study changes in blood-brain barrier permeability and transport elicited by pathological factors using in vitro models of the blood-brain barrier to better understand the molecular mechanisms and to find therapeutical approaches for bloodbrain barrier dysfunctions and (ii) to modulate blood-brain barrier functions for drug delivery to brain.

Effect of water structure on protein conformation

Group: Laboratory of Membrane Bioenergetics
Supervisor: Andras DÉR

The main goal of the research project is to reveal how structural changes of water influence protein function and conformation, and vice versa: how the molecular and mesoscopic architecture of the biological interface presented to water (on the level of proteins, lipids, membranes) influences the organisation of adjacent water molecules. Experiments and theoretical studies are planned on various model systems.

Bioelectronics

Group: Laboratory of Membrane Bioenergetics
Supervisor: Andras DÉR

We are going to investigate the electric and optical properties of biological macromolecules (mainly proteins), and to study the opportunities of their possible optoelectronic applications. Special emphasis will be laid on relevant methodological developments.

Molecular basis of the blood-brain barrier function

Group: Laboratory of Molecular Neurobiology
Supervisor: István KRIZBAI

By forming a single cell layer lining the blood vessels of the brain, cerebral endothelial cells (CECs) constitute the principal component of the blood-brain barrier (BBB). Tight junctions (TJ) and adherens junctions (AJ) play a key role in the maintenance of the barrier function. Despite considerable experimental efforts the molecular organization and regulation of cerebral interendothelial junctions is less well understood. Our project is focused on the role of interendothelial junctions under pathological conditions. The experiments will be carried out on an in vitro model of the BBB based on the culture of cerebral endothelial cells. Using different molecular, biochemical and immunofluorescent techniques we will investigate the changes in the expression, localization, interaction and posttranslational modifications of the junctional proteins (occludin, claudins, ZO-1, ZO-2, cadherins, catenins) in CECs in response to pathological conditions like oxidative stress and inflammatory stimuli. Proteomic analysis will be applied to find endothelial proteins involved in these pathological processes. Furthermore, planned experiments are designed to elucidate the role of different signaling pathways, protein phosphorylation and proteolysis in the pathomechanism of endothelial dysfunction.

Role of the blood-brain barrier in brain metastasis formation

Group: Laboratory of Molecular Neurobiology
Supervisor: István KRIZBAI

Brain metastases of malignant tumors are life threatening pathologies with limited therapeutic options. Therefore suppressing or reducing the risk of metastasis formation could be one of the most effective approach in the therapeutic strategies besides surgical removal of the primary tumor. Since the CNS lacks a lymphatic system, the only possibility for cancer cells to reach the brain is via the blood stream. Metastatic cells invading the CNS thus have to pass the blood-brain barrier (BBB). The tumors giving CNS metastases with the highest frequency are the malignant melanoma, lung cancer and breast cancer. By using an in vitro model of the BBB the project is focused on the elucidation of molecular mechanisms by which cancer cells cross the BBB. Different molecular, biochemical and immunofluorescent techniques will be applied to elucidate the role of various signaling mechanisms, proteolytic enzymes and brain derived factors in the transmigration of metastatizing cancer cells. Applicants interested in disease oriented basic biomedical research with affinity to modern molecular and biochemical techniques are welcome.

Investigation of biomolecules with optical micromanipulation

Group: Laboratory of Membrane Bioenergetics
Supervisor: Pál ORMOS

Using optical manipulation it is possible to manipulate single molecules, determine their mechanical properties. In optical traps formed by polarized light, it is possible to rotate the manipulated object, we can exert and measure mechanical torque. In the offered topics we investigate the torsional properties of biological macromolecules (DNA, actin, myosin), and we study interactions accompanied by molecular rotation.

Extension of the capabilities of optical traps by test objects of special shape

Group: Laboratory of Membrane Bioenergetics
Supervisor: Pál ORMOS

In optical tweezers generally spherical objects are manipulated, their location is controlled. If the manipulated object has a special shape, we can control its position, we can set its orientation, we can rotate it. We produce such objects by two photon excitation photopolymerization.. The topics covers photopolymerization induced by two photon excitation, and manipulaton of different objects of special shape.

Development of light controlled microfluidics devices

Group: Laboratory of Membrane Bioenergetics
Supervisor: Pál ORMOS

Microfluidics is the method of modern analytical biochemistry: the chemical processes take place in micrometer sized vessels, reactors. Due to the small size the systems behave in a specific manner, e.g. the Reynolds number characterizing the flow is very small, therefore the flow is always laminar, etc. Controlling the systems requires new methods. There is no concensus method in this area. In the offered topics we study fluid flow in microchannels, and explore how it is possible to control the complex systems by light.

Particle manipulation with optical tweezers

Group: Laboratory of Membrane Bioenergetics
Supervisor: Pál ORMOS

Studies involving single biological macromolecules, cells. We generate microscopic machines by photopolymerization, drive them by light, and use them to manipulate biological systems mechanically. At the moment we study torsional properties of DNA, and related DNA protein interactions.

Membrane protein folding - physical and molecular modeling

Group: Laboratory of Membrane Structure and Dynamics
Supervisor: Tibor PÁLI

Membrane proteins are under-represented among known atomic protein structures because it is difficult to crystallize and solubilize them in a folded form, which is, however, needed for standard structure biology approaches. The great interest, also of the pharmaceutical and cosmetics industry, in membrane protein structures demands alternative approaches. Based on our experience on membrane proteins, spectroscopic techniques and molecular modeling, we will develop a unique approach that yields simplified membrane protein structures, folds, constrained by spectroscopic data on the target proteins in their membrane environment. We have done this already "manually". We will automate the generation and selection of the best folds. This will be done by developing force fields and mechanics in a reduced protein representation. The method warrants functionally relevant predicted folds. The algorithm will be first developed from structural and kinetic data on selected representatives of trans-membrane alphahelix, beta-barrel, and water-soluble proteins, which are functionally or structurally bound to biomembranes, and generalized further by structural data from the literature.

Selected publications:
Kostrzewa, A., Pali, T., Froncisz, W. and Marsh, D. (2000) Membrane location of spin-labeled cytochrome c determined by paramagnetic relaxation agents. Biochemistry 39(20), 6066-6074.

Bashtovyy, D., Marsh, D., Hemminga, H.M. and Pali, T. (2001) Constrained modelling of spin-labelled major coat protein mutants from M13 bacteriophage in a phospholipid bilayer. Protein Science 10(5), 979-987.

Bashtovyy, D., Berczi, A., Asard, H. and Pali, T. (2003) Structure prediction for the di-heme cytochrome b-561 protein family. Protoplasma 221(1-2), 31-40.

Marsh, D. and Pali, T. (2004) The protein-lipid interface: perspectives from magnetic resonance and crystal structures Biochimica et Biophysica Acta - Biomembranes 1666(1-2), 118-141. (review article)

Pali, T., Bashtovyy, D. and Marsh, D. (2006) Stoichiometry of lipid interactions with transmembrane proteins - deduced from the 3-D structures. Protein Science 15(5), 1153-1161.

Biophysical studies on the vacuolar proton-ATPase (V-ATPase)

Group: Laboratory of Membrane Structure and Dynamics
Supervisor: Tibor PÁLI

The V-ATPase is a biomembrane-bound molecular rotary engine, which converts the chemical energy from ATP hydrolysis to the rotation of the rotor domain via a torque between specific subunits. This leads to trans-membrane proton pumping in the interface between the stator and rotor domains. The V-ATPase plays an important role in diseases like osteoporosis, acidosis and in the metastasis of tumors. Specific inhibition of certain sub-classes of the V-ATPase family has, therefore, direct medical and pharmaceutical relevance. To date there is no atomic resolution structure of the V-ATPase known and its mechanism of function is not known either. The primary long-term objective is the better understanding of structure-function relationship and the identification of functionally relevant structural changes in the engine. This project is aimed at the study of the proton pumping and ATP hydrolyzing functions of the Vo and V1 domains, respectively, their connection and interaction; the arrangement and interactions, also with lipids, of the Vo subunits; the rotation of the rotor domain; structural stability and the effect of structural and functional agents, e.g. specific inhibitors, on all these features in intact vacuoles, vacuolar vesicles and in reconstituted V-ATPase-lipid vesicles. We also aim to develop the structural models of the intra-membranous a and c subunits based on our structural data, whereas the function will be interpreted in physical models.

Selected publications:
Pali, T., Finbow, E.M. and Marsh, D. (1999) Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR. Biochemistry 38(43), 14311-14319.

Holzenburg, A., Jones, P.C., Franklin, T., Pali, T., Heimburg, T., Marsh, D., Findlay, J.B.C., and Finbow, M.E. (1993) Evidence for a common structure for a class of membrane channels. European Journal of Biochemistry 213(1), 21-30.

Pali, T., Finbow, E.M. and Marsh, D. (1999) Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR. Biochemistry 38(43), 14311-14319.

Kota, Z., Pali, T., Dixon, N., Kee, T.P., Harrison, M.A., Findlay, J.B.C., Finbow, M.E. and Marsh, D. (2008) Incorporation of transmembrane peptides from the vacuolar H+-ATPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarized infrared spectroscopy. Biochemistry 47(12), 3937–3949.

Measurement, analysis and simulation of spectroscopic data on biomembranes and membrane proteins

Group: Laboratory of Membrane Structure and Dynamics
Supervisor: Zoltán KÓTA, Tibor PÁLI

Spin label electron paramagnetic resonance (EPR), Fouriertransform infrared (FTIR) and fluorescence spectroscopy are complementary techniques, as they yield different types of structural data on biomembranes and membrane proteins. The interpretation of the com - bined data requires detailed spectral analysis. Our aim is to develop combined EPRFTIR and EPR-fluorescence experimental techniques for the simultaneous measurement of complementary structural data, on biomembranes and membrane proteins, and also to program suitable spectrum fitting algorithms, in order to obtain more details on structure and dynamics than what is currently available. The new techniques are to be first de - veloped on data on artificial, model membranes.

Selected publications:
Pali, T. and Marsh, D. (2001) Tilt, twist and coiling in beta-barrel membrane proteins: relation to infrared dichroism. Biophysical Journal 80(6), 2789-2797.

Marsh, D. and Pali, T. (2001) Infrared dichroism from the x-ray structure of bacteriorhodopsin. Biophysical Journal 80(1), 305-312.

Pali, T., Garab, G., Horvath, L.I. and Kota, Z. (2003) Functional significance of the lipid-protein interface in photosynthetic membranes. Cellular Molecular Life Sciences 60(8), 1591-1606. (review article)

Kota, Z., Pali, T. and Marsh, D. (2004) Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized ATR infrared spectroscopy and spin-label electron spin resonance. Biophysical Journal 86(3), 1521-1531.

Development of combined spectroscopic techniques for detecting the inverse hexagonal phase in biomembranes

Group: Laboratory of Membrane Structure and Dynamics
Supervisor: Zoltán KÓTA, Tibor PÁLI

A special class of lipids, which do not form bilayers in aqueous dispersions, play an important role in certain types of biomembranes. These lipids form an inverted hexagonal (HII) phase when dispersed in water. In biological membranes, which can have hundreds of different lipids, non-lamellar lipids mix with the "normal" lamellar lipids. The non-lamellar lipids appear to be important structurally for certain proteins and biological functions. The aim of the project is to use a number of different techniques (electron paramagnetic resonance, infrared and fluorescence spectroscopyidididifferen - tial calorimetry) and develop a combined approach, which will be able to detect the pres - ence of the HII phase and the lamellar-non-lamellar transition not only in model, but also in native biomembranes. Currently, there exist no reliable technique for this task.

Selected publications:
Kota, Z., Szalontai, B., Droppa, M., Horvath, G. and Pali, T. (1999) Fourier transform infrared and electron paramagnetic resonance spectroscopic studies of thylakoid membranes. Journal of Molecular Structure 481, 395-400.

Kota, Z., Szalontai, B., Droppa, M., Horvath, G. and Pali, T. (2002) The formation of an inverted hexagonal phase from thylakoid membranes upon heating. Cellular and Molecular Biology Letters 7(1), 126-128.

Kota, Z., Horvath, L.I., Droppa, M., Horvath, G., Farkas, T., and Pali, T. (2002) Protein assembly and heat stability in developing thylakoid membranes during greening. Proc. Natl. Acad. Sci. USA 99(19), 12149-12154.

Free radicals invading the human body - a spin trapping study

Group: Laboratory of Membrane Structure and Dynamics
Supervisor: Tibor PÁLI

Free radicals will be detected that enter the human body from food and by breathing. The technique will be spin-trapping electron paramagnetic resonance (EPR) because it is the most sensitive technique to detect and quantitate free radicals. In comparative stud - ies, we will identify different classes of free radicals and quantitate them in conserved dry food products and also in non-natural gases (cigarette smoke, car exhaust), to which humans are exposed. The results will useful for improving food conservation and gas fil - tering techniques.

Selected publications:
Nedeianu, S. and Pali, T. (2002) EPR spectroscopy of common nitric oxide - spin trap complexes. Cellular and Molecular Biology Letters 7(1), 142-143.

Kispeter, J., Bajusz-Kabók, K., Fekete, M., Szabó, G., Fodor, E. and Pali, T. (2003) Changes induced in spice paprika powder by treatment with ionising radiation and saturated steam. Radiation Physics and Chemistry 68(5), 893-900.

Kispeter, J., Fekete, M., Feher, L., Fodor, E., Kovacs, L., Laszlo, Zs. and Pali, T. (2004) Quality changes in dry onion products during conservation: A comparative study. Academic and Applied Research in Military Science 3(5), 689-693.

Nedeianu, S., Pali, T. and Marsh, D. (2004) Membrane penetration of nitric oxide and its donor Snitroso- N-acetylpenicillamine: a spin-label electron paramagnetic resonance spectroscopic study. Biochimica et Biophyisica Acta - Biomembranes 1661(2), 135-143.

Cellular and molecular biology of hormone induced neuro-glial plasticity

Group: Laboratory of Molecular Neurobiology
Supervisor: Árpád PÁRDUCZ

It is well documented that gonadal steroids exert organizational effects on developing neuronal connectivity and induce plastic changes on neuronal contacts in adults. Such effects are involved in the generation of sexually differentiated behavior and neuroendocrine functions. This project is focused on the study of cellular and molecular events related to synaptogenesis and synaptic plasticity in the sexually dimorphic areas of the central nervous system.

(1) the identification of estrogen-sensitive synaptic contacts in certain brain areas and their responses to estrogen during normal development and function.

(2) the identification of neuronal membrane components mediating the synaptic effects of sex steroids.

The role of gonadal hormones and neurosteroids in the neuroprotection

Group: Laboratory of Molecular Neurobiology
Supervisor: Árpád PÁRDUCZ

The objective of the proposed project is to investigate the role of sex hormones and neurosteroids in age-related changes of the nervous system and to explore their potential therapeutic value for reversing age-associated cognitive deficits. This program requires a multidisciplinary approach that spans from morphological, electrophysiological and behavioral studies to genomic analysis.
The proposed approach is original in a sense that it is based on recent observations of age-related dysfunctions in the nervous system being reversible and that the aging brain retains its capacity for plasticity and regeneration. An important aspect of neuronal plasticity is synaptic remodeling that could be a significant component in the regenerative capacity of the central nervous system (CNS). Research carried out in our laboratory demonstrates that among other factors, gonadal steroids are able to induce morphological synaptic plasticity in different parts of the CNS; and we have shown that dehydroepiandrosterone (DHEA) has similar morphogenic effects in both hypothalamus and hippocampus.

Role of impaired intracellular calcium homeostasis during neuronal degeneration

Group: Laboratory of Molecular Neurobiology
Supervisor: László SIKLÓS

Calcium ions play a second messenger role in nerve cells, thus their intracellular concentration is tightly controlled. If the regulatory mechanisms are impaired, several destructive processes are activated most of them are interconnected such a way that elevated intracellular calcium provides a positive feedback. Factors influencing the calcium equilibrium within nerve cells are the plasma membrane calcium channels and transporters, intracellular calcium storing/sequestrating organelles and intracellular calcium binding proteins, which may play different roles in different types of neurons and in different types of injury. The characterization of the calcium-mediated injury in different acute and chronic stress conditions (in models of acute injury and chronic degeneration) and attempts of stabilization of the calcium homeostasis in these paradigms are the aims of the present study.

Immune/inflammatory reactions during acute neuronal lesion and chronic neuronal degeneration

Group: Laboratory of Molecular Neurobiology
Supervisor: László SIKLÓS

The immune privilege of central nervous system (CNS) has been considered as experimentally well defined, however, recent data radically altered this viewpoint: peripheral immune cells can cross the intact blood-brain barrier, neurons and glial cells actively regulate macrophage and lymphocyte response, and there are immuno-competent cells in the CNS, such as microglial cells. Microglial cells even in minor pathological events start to proliferate, and while their primary aim is to eliminate local injury, infection, etc. – via components of oxidative burst – may considerably contribute to the degeneration. Thus, these cells act as a double-edged sword. In the present study, glial reaction next to the injured neurons are intended to be characterized in acute neuronal lesion or in conditions mimicking chronic degeneration to determine possible correlations between the degree of neuronal injury and the magnitude of local glial reactions.

Lipid-protein interactions in biological membranes

Group: Laboratory of Membrane Structure and Dynamics
Supervisor: Balázs SZALONTAI

Maintaining the barrier properties and the functioning (energy production, signal transduction, material transport etc.) of a biological membrane, a given membrane dynamics is required. This dynamics is adapted to the physiological conditions (e.g. temperature, light in photosynthetic organisms, different stresses) of the given membrane. Concerning barrier properties, the dynamics of membrane lipids and lipidprotein interactions are thought to be more important. The biological functioning is assured by the dynamics of the membrane proteins, which may depend also on lipidprotein interactions. The balance between lipid- and protein-dynamics and the role of lipid-protein interaction in maintaining membrane functionality is still not fully understood.
While there are several methods to study the structure and the dynamics of biologically relevant molecules – the applicant will use primarily Fourier transform infrared spectroscopy as studying method – in spite of their long history, not enough attention was paid to the lipid-protein ratios, lipid specificity and to their effect on the structure and the dynamics of the membrane/fragments. Since membranes are more and more considered as complicated assemblies of different domains, which might have fairly different properties, it would be very important to know precisely, not only the dynamics of the lipids, proteins and lipid-protein interactions in a membrane preparation but the relationship between the preparation methods and the obtained membrane features as well. Nowadays, when from many functional units of widely different functions of the membranes turns out that they are organised into lipid-rafts, where specific lipid composition helps to perform the biological function, such lipidprotein interaction studies are even more important. Special attention will be paid for these lipid-rafts.
For the work, several biological membranes will be used, thylakoid and plasma membranes of different cyanobacteria (where the lipid composition can be varied by physiology and genetic engineering), membranes of yeast cells, and mammals as well. These membranes will be prepared in cooperation with other groups. When needed, for more specific information, model systems will also be used. The spectroscopic techniques will include different methods of Fourier transform infrared spectroscopy (transmission, attenuated total reflection (ATR), surface enhanced infrared absorption (SEIRA), in combination with electrochemistry, orientation measurements etc.).
The candidate should be able to participate not only in the spectroscopic work but also in the preparation of the membranes as well.
Physicists, chemists or biologists (with interest in spectroscopy) are welcome.

Characterization of states and processes of the nervous system by video-pupillometry

Group: Laboratory of Membrane Bioenergetics
Supervisor: Zsolt TOKAJI

The importance of the information content of human movements (frequently representing deeper, e.g. molecular or even intramolecular processes as well) have long been known intuitively. (Think about e.g. the effects of ethanol contained in alcoholic drinks). However, methods that are really suitable for the quantitative study of these motions have either been developed only recently, or are now in the process of being developed. A movement, that is practically uncontrollable voluntarily, is the continuous change in pupil size. Pupil size is controlled by the nervous system, and depending on the experimental conditions, it reflects cognitive or emotional processes, sleepiness, the equilibrium of the sympathetic/parasympathetic nervous activities, or the effect of a drug or a treatment. Continuous measurement of pupil size can be performed by video-pupillometry the duration of which is usually between 1 and 15 minutes. Pupil records are rich in information, but the presently known methods extract from this only a very small fraction. Thus an aim of the study is to improve the video-pupillometric method by developing and testing new evaluation/interpretation techniques by using data sets that has been collected previously. Parallel to this, the experimental part consists of continuation of data collection mainly in the field of objective and quantitative characterization of presently mostly subjectively determined states such as sleepiness, anxiety, depression, or hyperactivity, and the effect of some physical/biological factors that could be beneficial in these states (such as bright light, darkness, or high density of negative air ions).

The role of the protein in the biological electron transfer processes

Group: Laboratory of Metalloprotein Biophysics
Supervisor: László ZIMÁNYI

Biological energy transformation is based on the migration of electrons through well organized protein chains towards lower energy. There is, however, a basic difference between the mechanism of electronic conductance in a metal wire and in a protein. In proteins, electron conductance is accomplished via quantum mechanical tunneling, which strongly depends on the intervening medium between electron donor and acceptor. Our goal is to better understand the mechanism of electronic conductance in proteins, and to study whether or not nature has optimized (and how) the conductance in certain important proteins. Our protein of choice is cytochrome c, the mobile electron carrier connecting complexes III and IV in mitochondria. We investigate intraprotein and interprotein electron transfer initiated by a covalent photoactive redox label attached to the protein surface. We study the effect of the structure and dynamics of the protein matrix on the rate of electron transfer. By producing mutants we map the electron transfer efficiency of the different regions of the protein. Kinetic spectroscopic experiments to determine the electron transfer rate and efficiency are supported by molecular modelling and electron transfer pathway calculations. Cytochrome c itself is a promising candidate as a component of biomolecule-based sensors, bioelectronic designs, so the understanding of its electric conductance is a high priority. Within the framework of an international collaboration we also plan to incorporate cytochrome c into hybrid biophotonic architectures – photonic crystals based on porous silicon. Thereby we expect to tune the optical properties of the semiconductor-based photonic crystals using the colored cytochrome protein. Conversely, we intend to study the effects of the interesting nonlinear optical phenomena characteristic of the photonic crystals on the optical properties of the protein.

The mechanism of the maturation of cytochrome c

Group: Laboratory of Metalloprotein Biophysics
Supervisor: László ZIMÁNYI

Cytochromes are heme containing proteins, which carry out diverse physiological functions, such as electron transfer, an important process in the energy metabolism of living cells. C type cytochromes are distinguished from other cytochromes by the covalent attachment of the heme group to the protein. The advantages of this covalent binding are so far unclear. The maturation of mitochondrial cytochrome c, i.e. the covalent binding of the heme cofactor, is catalysed by the enzyme cytochrome c heme lyase. Despite the importance and the widespread occurence of these proteins from yeast to human, we know little about the catalytic process or about the interaction of the two proteins and the heme. Indirect information about this interaction is supplied by our observation of spontaneous cytochrome c maturation (covalent heme attachment in the absence of the cytochrome c heme lyase) at a much lower efficiency than in the presence of the heme lyase. However, the spontaneously matured cytochrome c has slightly different physico-chemical parameters than the native protein. This indicates that the function of the heme lyase is not only the catalysis (acceleration) of the covalent heme attachment, but also to facilitate the formation of the final, native conformation (structure) of cytochrome c.
We study by spectroscopic methods the function, and the interaction with its substrates, of the heme lyase. The enzyme is assumed to be at least partially intrinsically unstructured, and to fold into transitional structures potentially, upon the interaction with heme and apocytochrome, thereby promoting the formation of the covalent bond. Site directed mutagenesis and heterologous expression of the heme lyase, followed by in vitro cytochrome c maturation experiments is expected to yield information on the role of typical sequence motifs in the maturation process.