National Repository of Grey Literature 6 records found  Search took 0.00 seconds. 
Mass spectrometry analysis of integral membrane peptides
Sabó, Ján ; Cebecauer, Marek (advisor) ; Pompach, Petr (referee)
Biological membranes ensure a large scale of vital processes in the living organisms. Lipid and protein composition is very complex in the membranes. Therefore, simplified model systems were developed to study basic mechanisms regulating the function of membranes. To simulate transmembrane proteins of the type I and II, the short, α-helix-forming, synthetic peptides are employed. The hydrophobic character of the peptides and their transbilayer positioning in membranes well represents transmembrane domains of proteins in the living organisms. One of the simplest and most widely used model membrane systems are liposomes. Methods for their formation has been known for a long time. Quantification of their components after the process of liposome preparation is challenging, but for the maximal control over given model system very desirable. In this work, we adapted a formerly described protocol for delipidisation of the transmembrane peptides for their consequent characterisation by LC/MS. A relative amount of peptide successfully incorporated into vesicles was acquired by the analysis of extracted chromatograms of peptide ions. We demonstrate that analysis of vesicles with peptide content of 5-10 mol% is feasible and the loss of the peptide is below 50 %. Such vesicles can be used for further...
The effect of peptides derived from protein transmembrane domains on membranes
Olšinová, Marie ; Cebecauer, Marek (advisor) ; Obšil, Tomáš (referee) ; Vácha, Robert (referee)
Rich structure of cell membranes raises broad number of questions regarding the mechanisms driving and regulating processes taking place on membranes. The thesis presents four articles investigating organization of lipid membranes and peptide-lipid interactions. The experiments were performed on model lipid membranes. These simplified systems that partially mimic cell membranes enable to study protein-lipid interactions at the molecular level and membrane physico-chemical properties in a controlled way. Advanced fluorescence techniques such as FCS, TDFS, FLIM and anisotropy were used for the system characterization. First publication describes newly designed fluorescence dyes based on boron dipyrromethene structure, the so- called molecular rotors, which are reported to be viscosity-sensitive probes. Detailed analysis of fluorescence lifetime of excited state of the molecular rotors inserted into lipid membranes showed diverse incorporation of dyes into membranes and their reorientation in membranes of different rigidity. The second part investigates existence of lipid nanodomains in membranes caused by the presence of a cross-linker. Even though standard fluorescence microscopy techniques do not allow direct visualization of the nanodomains, we were able to detect these structures by employment of...
Characterization of native and heterologously expressed membrane transporters in yeast using fluorescent probes
Zahumenský, Jakub ; Gášková, Dana (advisor) ; Cebecauer, Marek (referee) ; Krůšek, Jan (referee)
Yeast plasma membrane transporters play crucial roles in many cellular processes, including detoxification and build-up and maintenance of the plasma membrane potential (ΔΨ). The former development of the diS-C3(3) fluorescence assay by the Biophysics Group of the Institute of Physics, Charles University, enabled us to conveniently study both, including their changes, using a simple fluorescent probe diS-C3(3). Many studies carried out on both animal and yeast cells have revealed that ethanol and other alcohols inhibit the functions of various membrane channels, receptors and solute transport proteins, and a direct interaction of alcohols with these membrane proteins has been proposed. Using the diS- C3(3) assay for multidrug-resistance pump inhibitors in a set of isogenic yeast pdr5 and snq2 deletion mutants we found that n-alcohols (from ethanol to hexanol) exhibit an inhibitory effect on both pumps, increasing with the length of the alcohol carbon chain. The inhibition is not connected with loss of plasma membrane structural or functional integrity and is fully reversible. This supports a notion that the inhibitory action does not necessarily involve only changes in the lipid matrix of the membrane but may entail a direct interaction of the alcohols with the pump proteins. Tok1p is a highly specific...
Impact of protein transmembrane domains on membrane organisation
Šljivnjak, Erna ; Cebecauer, Marek (advisor) ; Novotný, Marian (referee)
Plasma membrane is not a static, but rather a dynamic structure that constantly changes its form and local properties. Interactions between building blocks of plasma membrane and external factors are responsible for those changes. In this thesis, I summarize the literature which describes interactions between transmembrane proteins and lipid membranes as well as its consequences. I discuss membrane thickness, respectively thinning, protein sorting and clustering shown to be dependent on the properties of transmembrane domains. Furthermore, the role of proteins in various model of the plasma membrane organization are indicated. Finally, I report on currently discovered impact of the surface roughness of TMDs on local mobility and organization of lipids. All these data indicate importance of detailed understanding of TMDs, their properties and relation to surrounding lipid membranes. Key words: membrane, protein, transmembrane domain, hydrophobic mismatch, protein sorting, models of the plasma membrane
Localisation of CD4 coreceptor and its variants in human T cells
Glatzová, Daniela ; Cebecauer, Marek (advisor) ; Drbal, Karel (referee)
CD4 co-receptor of main T cell receptor (TCR) is essential for proper development of T lymphocytes and their function in adaptive immune responses. It is believed that CD4 stabilizes the interaction of TCR with antigenic ligand, peptide-MHC, and thereby improves T cell-dependent responses during immune reaction. CD4 is transmembrane glycoprotein with a number of structural motifs in its intracellular domain which do not dramatically affect its sorting to the plasma membrane but can influence its local organization at nanoscale. CD4 was shown to transiently accumulate in the immunological synapse formed between T cell and antigen-presenting cell. Such accumulation is rapidly followed by its internalization and/or delocalization outside the synapse. This is in contrast with TCR which accumulates strongly in the immunological synapse and is later found enriched in the central area of this structure. It is therefore unclear how TCR and its CD4 co-receptor function together when binding to their common ligand during the initiation of signaling in T cells. We aim to study localization of CD4 at nanoscale using advanced fluorescence microscopy techniques achieving significant improvements in resolution. In this work, CD4 and its mutant variants, potentially causing its different localization at the...
Expression of the recombinant extracellular parts of leukocyte receptors AICL a NKR-P1CBALB
Čonka, Martin ; Novák, Petr (advisor) ; Cebecauer, Marek (referee)
v anglickém jazyce NK cells represent a population of lymphocytes which are able to kill certain tumor cells or virally infected cells. The subject of the diploma thesis is a mouse NK cell receptor mNKR- P1CBALB and a human leukocyte receptor hAICL. The mNKR-P1CBALB belongs to the activating receptors and is able to activate the cytotoxic functions of NK cells. The hAICL receptor is a ligand to the NKp80 which is an activating receptor of NK cells. Interaction between these two proteins leads to the activation of effector functions of NK cells as well. The aim of this work was the preparation of the recombinant extracellular parts of receptors mNKR-P1CBALB and hAICL, the optimalization of their in vitro refolding and the characterization of proteins using mass spectrometry. The proteins samples will be used for further structural study of the extracellular parts of these leukocytes receptors.

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