| dc.description.abstracteng | According 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
microscopy. | de |