Self-assembly and potassium ion triggered disruption of peptide-based soft structures

This report describes formation of soft vesicular structures by a tetrapeptide and its disruption triggered by potassium ions

Crystal Structure of the Minimalist Max-E47 Protein Chimera

Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5′-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47 dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts

Stability of cross-linked collagen

Teoretická část této bakalářské práce pojednává o obecných vlastnostech kolagenu, který je nejčastěji rozšířeným vláknitým proteinem v těle, o síťování kolagenu, které vede ke zlepšení mechanických i chemických vlastností, a o široce využívaných síťovacích činidlech, především 1-ethyl-3-(3-dimethylaminopropyl) karbodiimidu (EDC). Dále se zabývá několika metodami stanovení EDC ve vodném roztoku. Experimentální část je zaměřena na praktické stanovení množství EDC, které zůstalo ve vodě použité k promývání vzorků po síťování. Cílem bylo zjistit, jestli současně používaná metoda promývání je dostatečná pro jisté aplikace nebo je potřeba ji upravit. I přesto, že sloučenina EDC je prakticky netoxická, může způsobovat zbarvení kolagenních materiálů po sterilizaci ozařováním. Vzhledem k tomu, že nebylo zjištěno jaké množství EDC může ve vzorku zůstat nebo jestli je nezbytné vymýt veškeré EDC, je obtížné rozhodnout jestli je promývací metoda dostatečná nebo není. Navíc je potřeba brát do úvahy i ostatní vedlejší produkty síťování kolagenu. Nicméně cílem této práce bylo pouze zhodnotit účinnost vymývání EDC z kolagenních vzorků. Pro síťování byly použity tři různé koncentrace EDC a následně byly vzorky vymývány po různě dlouhou dobu. Takto bylo získáno 48 vodných vzorků, které byly následně třikrát spektrofotometricky stanoveny. Výsledně bylo zjištěno, že pro běžně používanou koncentraci síťovacího činidla (50 mmol.l 1 EDC) postačuje promývat vzorky po dobu 30 minut v roztoku hydrogenfosforečnanu sodného (Na2HPO3) a následně promývat ve vodě čtyřikrát po dobu 15 minut s výměnou vody při každém kroku. Po těchto krocích klesla koncentrace EDC v promývací vodě pod 1 mol . l-1, pro nižší koncentrace síťovacího roztoku byla dostatečná i kratší doba promývání. Výsledky této bakalářské práce jsou pouze předběžné, a proto by mělo následovat další zkoumání této problematiky.Theoretical part of this bachelor thesis is based on general information about collagen, most common fibrous protein in body, collagen cross-linking; leading to its better mechanical and chemical properties; and widely used crosslinking agents, mainly based on 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide (EDC). Furthermore it deals with several methods of quantifying EDC in aqueous solutions. Experimental part is concerned with the determination of EDC remained in water used for washing samples in order to purify them after the cross-linking. The goal is to find out if used washing method is adequate enough for certain application or if it needs some adjusting. Even if EDC is practically non-toxic, it provides side effects on colour of the collagenous material after the sterilization by irradiation. Since it has not been found in the literature what specific amount of EDC can stay in a sample or if it is necessary to be completely washed out. According the state-of-the-art, it is difficult to assessed if the washing method is sufficient or not. Moreover, there are also other side products of cross-linking collagen in the final collageneous products that have to be taken into account. However, the aim of this bachelor thesis is to evaluate washing EDC from the collagenous sample only. As for cross-linking method, three concentrations of EDC followed by washing samples for different periods of time were used. Following this method, 48 samples were prepared and three times measured using UV-VIS colorimetric assay. As a result it was found out that for commonly used concentration of cross-linking agent (50 mmol.l-1 of EDC) it is sufficient to wash samples for 30 min in solution of disodium phospnonate (Na2HPO3) followed by washing with water 4 times for 15 min with changing the water at each step. After these steps, the concentration of EDC in washing water was reduced up to 1 mol.l-1. For lower concentrations of cross-linking EDC solution it is sufficient even shorter washing time. Considering the preliminary results of this bachelor thesis, further investigation dealing with this topic should follow.

Functional studies of plasma membrane syntaxins in yeast

Syntaxins are required for fusion of membranes in eukaryotic cells and belong to a group of proteins known as t-SNAREs. This thesis primarily focuses on the role of the plasma membrane syntaxins Sso1p and Sso2p in the yeast Saccharomyces cerevisiae. The plasma membrane syntaxins are required for viability in yeast, but in the vegetatively growing cell, the Sso proteins have seemingly reduntant functions. We generated a mutant allele of SSO2, sso2-1, that has a conditional lethal phenotype in the absence of SSO1. Overexpression of genes coding for other SNARE proteins; Sec9p, Snc1p and Snc2p, suppressed the lethal phenotype. The corresponding mutant allele of SSO1, sso1-1, is also temperature-sensitive and interacts synthetically with a disruption of MSO1, which codes for a Sec1p interacting protein. Most notably, both SSO1 and MSO1, but not SSO2, were shown to be necessary for spore formation during meiosis. Mapping of functions within the Sso1p protein showed that a region in the N-terminus of Sso1p is needed for efficient sporulation. Unexpectedly, the 3’-untranslated region of SSO1 is absolutely required for sporulation and also sufficient to enable some spore formation when fused to the SSO2 open reading frame. Inspection of the sso1/sso1 phenotype during sporulation using transmission electron microscopy showed that prospore membrane assembly at the meiotic plaque of the spindle pole body is completely blocked in the mutant. A second part of this thesis deals with screening for uncharacterized genes involved in intracellular transport by exposing deletion mutants for drugs known to inhibit intracellular transport. The screen identified two new genes whose deletions made the cell sensitive to monensin, and those were given the names MON1 and MON2. Five new genes caused sensitivity to Brefeldin A when deleted, and were named BRE1-BRE5

pH-dependent coarse-grained model of peptides

We propose the first, to our knowledge, coarse-grained modeling strategy for peptides where the effect of changes of the pH can be efficiently described. The idea is based on modeling the effects of the pH value on the main driving interactions. We use reference data from atomistic simulations and experimental databases and transfer its main physical features to the coarse-grained resolution according the principle of "consistency across the scales". The coarse-grained model is refined by finding a set of parameters that, when applied to peptides with different sequences and experimental properties, reproduces the experimental and atomistic data of reference. We use the such parameterized model for performing several numerical tests to check its transferability to other systems and to prove the universality of the related modeling strategy. We have tried systems with rather different response to pH variations, showing a highly satisfactory performance of the model.Comment: accepted for publication in Soft Matte

Three-Dimensional Structure of the Complexin/SNARE Complex

During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca2+-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel α-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca2+-evoked neurotransmitter release

Long-period gratings in chalcogenide fibers

Les verres chalcogénures sont des matériaux optiques connus depuis 50 ans. Ils sont fortement non-linéaires, transparents dans l'infrarouge et photosensibles à la lumière visible. Grâce à des percées récentes du côté des procédés de fabrication, leurs applications dans divers domaines sont devenus possibles, notamment dans les capteurs biochimiques, le guidage de sources infrarouges de haute puissance, la fabrication de sources infrarouges et dans les bascules optiques. Ce mémoire présente les résultats de travaux de recherche réalisés sur les fibres chalcogénures de AS₂S₃, notamment dans la conception de réseaux à longs pas (LPG), photo-induits et mécaniquement induits. Premièrement, une revue bibliographique présentant les propriétés et les applications des fibres en verre chalcogénures est fournie. Ensuite, une brève introduction sur la théorie des fibres optiques et des réseaux LPG est développée, pour finalement présenter les résultats expérimentaux. Ces derniers portent sur les observations des effets photo-induits, et sur la fabrication et la caractérisation, à la fois, des réseaux LPG mécaniquement induits, des interféromètres Mach-Zehnder utilisant deux réseaux LPG en série, et d'un réseau LPG photo-induit