Thermodynamics and structure of peptide-aggregates at membrane surfaces

This thesis aimed at improving our understanding of the thermodynamic and structural aspects of peptide aggregation processes at membrane surfaces. For this purpose we investigated a class of model peptides, which form a [beta]-sheet structure upon binding to membrane surfaces. Binding of peptides with the repeating sequence of KIGAKI to anionic membrane surfaces was chosen as model system to characterize the transition from a random coil to [beta]-sheet structure. Evidence is brought that the process of intermolecular [beta]-sheets formation by the KIGAKI peptides is a suitable model system for a peptide aggregation process at membrane surfaces. In order to understand this aggregation process, thermodynamic parameters of (KIGAKI)3 binding to lipid membranes were determined directly by isothermal titration calorimetry. For a description of the peptide binding data a theoretical binding model was developed and evaluated with the drug verapamil. It is shown that the binding model, which is based on the Gouy-Chapman theory, can be used in a general way to describe electrostatic attraction and repulsion of charged molecules to lipid membranes under a variety of environmental conditions. Nevertheless, binding of peptides to lipid membranes is more complex as simply considering electrostatic attraction of the peptide to the membrane. Thermodynamic binding parameters of (KIGAKI)3 to lipid membranes, obtained by ITC, combines mainly two reactions, the intrinsic binding and [beta]-sheet folding process. Separation of both subprocesses from the overall thermodynamic binding process could be achieved by varying the extent of [beta]-sheet formation due to substitution of two adjacent D amino acids within the peptide sequence. Double D amino acid substitution leads to a local disturbance of the [beta]-sheet structure, where the extent of the [beta]-sheet formation is dependent on the number and position of the double D amino acid substitution. With this approach it was possible to determine for the first time a full thermodynamic profile of the random coil to [beta]-sheet transition for a peptide in a membrane environment and concomitantly these parameters are the first clearly defined parameters of a peptide aggregation reaction. Beta sheet folds in proteins tend to be distinctively smaller than current models predict for [beta]-sheets in protein and peptide aggregates. To reveal differences between the [beta]-sheet folding reaction in a native and aggregated protein, we extended the study and determined the length dependence of the [beta]-sheet folding reaction. Thermodynamic parameters of the [beta]-sheet folding reaction for KIGAKI peptide with different lengths were determined in analogy to (KIGAKI)3. A linear length stabilization effect could be demonstrated for KIGAKI [beta]-sheet structure. Furthermore, for [beta]-sheets shorter than 10 residues the folding reaction is driven by entropy, whereas for longer [beta]-sheets the folding reaction is driven by enthalpy. Underlying length dependence of the thermodynamic driving forces of [beta]-sheet folding reaction is therefore the most important finding of this work since it reveals an important difference in the folding reaction between native and aggregating [beta]-sheets. Furthermore, the double D amino acid substitution strategy opens a new way to systematically resolve the characteristic [beta]-sheet-aggregation at membrane surfaces, as for example for the Alzheimer peptide. Beside thermodynamics of the [beta]-sheet folding process we also studied the dynamics and size of the extended [beta]-sheet structure of KIGAKI at the membrane surface by deuterium solid state NMR. It is revealed that the [beta]-sheet structure formed by the (KIGAKI)3 peptide is large and rigid and therefore inevitably extended at the membrane surface. Perturbation of the membrane integrity, due to peptide binding and [beta]-sheet formation are not observed. In turn, these findings weaken the theories that peptide aggregates at the membrane surface mediating cell death by disrupting the cell membrane. As a new approach to study extended [beta]-sheet structures at membrane surfaces, the (KIGAKI)3 and Alzheimer peptide were encapsulated in reverse micelles and dissolved in a low viscosity solvent. Within the reverse micelles KIGAKI peptides adopted their characteristic [beta]-sheet structure. Promising NMR results show that the reverse micelles technique is an interesting alternative for the structure analysis of membrane peptide and protein aggregates. The last part of this thesis dealt with the partitioning process of xenon in lipid membranes. NMR spectroscopic analysis of the chemical shift behavior of 129Xe in lipid suspension offered a new method to determine partitioning coefficients of xenon in lipid membrane samples, like blood and tissue samples, which are of particular interest for various medical applications of xenon. Additionally, our data provide new aspects of the anesthetic properties of xenon. In particular, we demonstrated that lipid molecules maintained their structure upon xenon partitioning, which suggests that structural changes of the lipid molecules are not necessary to mediate anesthesia.