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Study of factors influencing proton transport mediated by MntH protein.
Straková, Eva ; Urbánková, Eva (advisor) ; Žáčková, Markéta (referee)
The natural resistance-associated macrophage proteins (Nramp) form a family of secondary active transporters facilitating the transport of divalent metal ions (Mn2+, Fe2+, Cd2+, Co2+, Zn2+) across biomembranes. Bacterial proteins belonging to the Nramp family were describedas proton-dependent manganese transporters - MntH. MntH homolog from E. coli representing a model system for structure function relationship was used in this study. Metalinduced proton transport mediated by MntH was measured by means of changes in intracellular proton concentrations using pH sensitive fluorescent protein pHluorin. This approach does not allow proper estimation of real quantity of transported protons, since the internal pH is tightly regulated. The cellular buffering capacity was calculated from measured pH changes induced by addition of propanoic acid, in order to better quantify the transport. The changes in [H+] induced by cadmium were recalculated using buffering capacity for the wild type MntH and its single-point mutation N401G, known to influence intracellular pH. It was shown that the chosen approach significantly influences results, including the dependence of transport on external pH. Potential influence of cadmium to respiration was imitated by using inhibitors of respiratory chain (KCN, NaN3) in measurements...
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Intracellular pH of yeast and its regulation by transport of potassium ions
Hošková, Barbora ; Urbánková, Eva (advisor) ; Holoubek, Aleš (referee)
Cytosolic pH of yeast is regulated and maintained by cooperation of many transport proteins. A fluorescent protein pHluorin was used in this thesis to evaluate cytosolic pH of yeast Saccharomyces cerevisiae. pHluorin is sensitive to pH of its surrounding. The experiments were mostly carried out using spectrofluorometer, however also a method which enables using reader was improved and optimalized for future use. As well a specific arginin-phosphate growth medium was used. An influence of certain proteins on the ability to regulate cytosolic pH was also studied. Some proteins are connected with the regulation directly e. g. Pma1 ATPase, some of them in an indirect way e. g. Ypt6. It was proved, that the value of cytosolic pH depends on yeast strain and cultivation and experimental conditions. None of the gene deletions studied in this thesis changed regulation of pHcyt much, the cells were able to adjust pHcyt even in situation, when the external medium is acidified by sulphuric acid. Buffering capacity was measured with addition of weak base to the cell suspension. The values varied in range from 30 to 60 mM if the cells were cultivated in YNB growth medium. Cells cultivated in arginin-phosphate medium had higher buffering capacity.
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Study of factors influencing the function of MntH, membrane transport protein of E. coli
Jurková, Ingrid ; Urbánková, Eva (advisor) ; Krůšek, Jan (referee)
MntH belongs to the Nramp family of transport proteins, and plays an important role not only in homeostasis of iron and manganese, but also in bacterial defence against the immunity response of an infected host cell. MntH co-transports divalent metal ions into the cell together with protons with a stoichiometry dependent on the membrane potential and extracellular pH. Using the redistribution potential dye diS-C3(3), we measured the effects of MNTH expression and MntH-mediated metal transport on the cell membrane potential and intracellular pH. Cells expressing MNTH were found to be hyperpolarised and their membrane potential was depolarised upon the addition of metal ions. In the theoretical part of our work, we explored general four-, six-, and eight-state carrier models that were modified by introducing the voltage dependence of all rate constants. Using mathematical modelling, we simulated the effect of various model parameters (including membrane potential, substrate concentration, and carrier or substrate charge) on substrate influx. We observed some of the transport characteristics described for MntH proteins such as variable symport stoichiometry that is influenced by the membrane potential and pH. However, for a more detailed simulation of the eight-state carrier model, more information about...
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Study of the relations between the structure and function of C - terminal vanilloid receptor TRPV1
Gryčová, Lenka ; Obšil, Tomáš (advisor) ; Heřman, Petr (referee) ; Urbánková, Eva (referee) ; Krůšek, Jan (referee)
Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labeling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations. Calmodulin (CaM) is known to play an important role in the regulation of TRP channels activity. Although it has been reported that CaM binds to the Cterminus of TRPV1 (TRPV1-CT), no classic CaM-binding motif was found in this region. In this work, we explored this unusual TRPV1 CaM-binding motif in detail and found that five residues from a putative CaM-binding motif are important for TRPV1-CT's binding to CaM, with arginine R785...
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