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Metal-induced aggregation of ß-peptides on membrane surfaces

dc.contributor.advisorAlcarazo, Manuel Prof. Dr.
dc.contributor.authorWiegand, Markus
dc.titleMetal-induced aggregation of ß-peptides on membrane surfacesde
dc.contributor.refereeAlcarazo, Manuel Prof. Dr.
dc.description.abstractengAccording to the liquid mosaic model of Singer and Nicolson, the cell membrane defines the boundaries of a cell to the inside and outside and describes a twodimensional lipid bilayer. Integrated into this lipid bilayer are various membrane proteins that are involved in a large number of vital processes and tasks. These tasks include signal transduction as well as transport into and out of the cell. These biomolecules can interact at the membrane surface or as a transmembrane domain with the membrane. In contrast to the hypothesis according to the liquid mosaic model, the peptides and proteins in a membrane are not completely unrestricted in their mobility along the membrane. In order to perform all membrane functions, a long-range molecular and structural organization is required. In order to ensure a long-range structural organization and stability, the so-called membrane skeleton (MSK) is involved as part of the cytoskeleton. The MSK forms and stabilizes domains in the membrane, provides the membrane with increased mechanical stability and provides important anchor points for various other cytoskeletal components. In order to explain the mechanical stabilization of the membrane by the MSK, the anchored protein picket model was established. According to this model, the MSK forms a kind of "net" that holds the components of a membrane together on one side and prevents them from diffusing freely through the membrane on the other side. It is still unclear how the MSK controls the domain formation and differentiates between the individual membrane components. An important aspect here is to improve the understanding of the interaction between the membrane (surface) and the membrane components with a main focus on the membrane-peptide interaction. In order to bring this highly complex issue to a more comprehensible level, various model systems have been developed and introduced. To reduce the complexity of the investigated system the peptidomimetics were developed. With the peptidomimetics it is possible to reduce the function of the used peptides and to analyse the basic properties and functions of this system. In the presented thesis β-peptides with their well defined secondary structure was used to mimic natural peptides that interact with the surface of a membrane to manipulate the composition of the membrane or to introduce a peptide network. Both aims should lead to a deeper insight in the lipid-peptide interaction and how lipid domains are formed. The model β-peptides were synthesized by using the Fmoc/tBu based solid-phase peptide synthesis (SPPS) with multiple instances of orthogonal protective groups. With this flexibility during the synthesis it was possible to get access to a toolbox-like synthetic strategy, what lead to an easy and fast way of synthesising different β-peptides. With this toolbox one peptide backbone was synthesized, which could be modified at the end with different recognition units or lipid anchors. Even the access to blind β-peptides as a negative control during the versatile analysis was simplified. Multiple generations of β-peptides were synthesized and with each generation the properties and functions were further optimized. The first generation of β-peptides P-49 (Fig. 6.2) was very hydrophobic and showed a strong tendency to be membrane active and therefore to disturb the membrane integrity and therefore the analysis. The advantage of this system was, that the binding abilities were very pronounced and P-49 bind very well to the surface of a membrane. In the following generations it was tried to optimize the binding abilities without the down side of disturbing the membrane integrity. The further β-peptides, like P-55, were modified so that they had a much more hydrophilic character. By introducing additional Asp amino acids, additional net charges were introduced into the β-peptide, which made the β-peptide much more soluble, especially in aqueous media, but the β-peptide also stopped binding to the membrane surface. Some of the more hydrophilic character was removed for the following generation (P-56, 57, 58 and 59). With this generation a good compromise could be found between the hydrophilic component to ensure solubility and the hydrophobic component so that the β-peptides can bind to the membrane. The first property analyzed property of the synthesized β-peptides was the secondary structure by CD-spectroscopy. The secondary structure is an important component of the whole thesis, without a well defined and known secondary structure the design and the corresponding analysis of the β-peptides would be worthless. For all analyzed β-peptides could be shown, that the β-peptides, no matter from which generation they come, all have a right-handed 14-helix as planned. Like expected their are differences in the intensity of the signals. In organic solvents the secondary structure is more pronounced, than in aqueous media. The organic solvents support the secondary structure and therefore an increased intensity could be observed. A main focus of this work was the analysis of the β-peptides with respect to the ability of a metal induced aggregation and to form a kind of peptide network on the surface, similar to the MSK or lipid domains in the membrane. To answer this question, first it was investigated the binding of metal ions by the β-peptides and their recognition unit(s) and then it was tried to transfer these results to the metal induced aggregation on model membranes. The coordination of metal ions by the β-peptides was investigated by UV/Vis titrations. This was done using the property of the recognition unit that the metal coordination significantly changes the absorption spectrum. Three different metal ions were analyzed and it could be shown that the β-peptides quantitatively coordinate the metal ions in solution. To get a deeper insight in the coordination itself, the Job-plot analysis was performed. It allowed to draw conclusions about the coordination number between the β-peptides and the metal ions. For some β-peptides higher aggregates, which indicate a network-like coordination, were found. It has to keep in mind, that the Job-plot method is not undisputed. For example, this method cannot differentiate between aggregates with the ratio 1:1, 2:2 or 3:3. All ratios that represent an integer multiple always give the same result. With these positive results in hand, the next step was tried. It should be tested, if a metal induced aggregation on a model membrane can be demonstrated. Before this can be done, it has to be checked, if the β-peptides bind to the surface of a model membrane. For this purpose a binding study was performed, where fluorescence labeled β-peptides should bind to a membrane, which were also labeled. If a FRET-effect could be observed the β-peptides are binding to the membrane. This experiment was performed with all synthesized β-peptides and the summary of these experiments is, that the β-peptides show different binding affinities on the surface of a membrane. The generation of β-peptides around P-50 show a strong affinity to bind to the surface. The next generation of β-peptides (P-55/P-62) showed no binding at all and the last generation represented by P-56, 57, 58 and 59 showed a combination of the two further mentioned generations. P-56, 57, 58 and 59 showed a good binding ability, the affinity was lower than for P-50, but higher than for P-56, 57, 58 and 59. Up to this point it could be shown that the β-peptides take on a defined secondary structure, that the β-peptides successfully bind metal ions in solution and also bind to the surface of membrane. The aim is to bring together these partial results in a further analysis. For this purpose the following experiment was constructed and carried out (Fig. 9.27). With this experiment, the previous experiments and their results should be combined with each other. Two labeled vesicle systems with unlabeled β-peptides were combined. During the experiments, a defined amount of metal salt solution was added and it was observed whether the β-peptides coordinate the metals accordingly, thus bringing the two vesicle systems in close proximity to each other to cause and observe a FRET-effect. Different variations of the vesicle diameter (100 – 400 nm) or different concentrations of lipid labeling (0.2 and 1.5 mol%) were tested for the experiments. For some combinations, a corresponding FRETeffect was observed after the metal salt solution was added. Also, for many of the experiments no FRET-effect could be observed, but a decrease in the intensity was always observed during the measurements. This observation could not be explained marked vesicles and in red (right side) the Texas Red™DHPE marked vesicles. directly and was therefore not to be expected in this context. A literature search confirmed the assumption that the β-peptides are membrane active and thus have disturbed the membrane integrity. To support this assumption with experimental results, a leakage assay was performed to determine the level of membrane activity and to compare the β-peptidesThis analysis showed, for example, that the first gen- ˙ eration of β-peptides disturbs the membrane integrity very strong and thus causes a very strong leakage. In the following generations, this behaviour was reduced, but partly at the expense of the interaction with the membrane. For example, the β-peptides P-50 no longer disturbed the membrane integrity, but also the β-peptides did not longer bound to the membrane. The last generation of β-peptides then showed a moderate behaviour. These β-peptides still attempted a leakage, which was significantly lower compared to the β-peptides of the first generation and they bound to the surface of the membrane. With this thesis, the first promising foundations for further research have been made. Metal coordination by the β-peptides in solution was successfully demonstrated, and it could also be shown that the β-peptides bind to the surface of a membrane. In further experiments it should now be shown that the β-peptides can bind the metal ions on a membrane surface and thus form a network-like system on the surface. A suitable method would be for example the atomic force
dc.contributor.coRefereeSteinem, Claudia Prof. Dr.
dc.contributor.thirdRefereeTittmann, Kai Prof. Dr.
dc.contributor.thirdRefereeKoszinowski, Konrad Prof. Dr.
dc.contributor.thirdRefereeEnderlein, Jörg Prof. Dr.
dc.contributor.thirdRefereeBennati, Marina Prof. Dr.
dc.subject.engMembrane surfacede
dc.subject.engLipid domainde
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de

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