The preferential adsorption to the lipid bilayer surface over peptide aggregate self-assembly was the key difference observed in the simulations of two model peptides with and without DMPC model membrane.
In both scenarios, the initial events of peptide adsorption were accompanied by a prominent reduction of nonpolar solvent accessible surface area. However, the large surface presented by the DMPC lipids accommodated the peptide side chains seemingly more efficiently than the small and compact oligomeric aggregates. In particular the partitioning of aliphatic side chains (Ala, Val, Leu, Ile) to the interfacial region and center of the lipid bilayer is energetically favorable as indicated by MD simulations with an umbrella sampling protocol [338]. These findings suggest a competition between peptide aggregate self-assembly and adsorption to the model membrane. Moreover, the solvent-exposed hydrophobic peptide surface in the presence of a DMPC bilayer was strongly decreased in comparison to the observed peptide oligomerization
without a DMPC bilayer. This result confirms the role of hSAS reduction (i.e. the hydrophobic effect) as a driving force for peptide aggregation.
Polar and charged head groups constitute the interfacial region of the DMPC lipids to the solvent and exhibited favorable coulombic interactions to the polar peptide residues, as well as the peptide main chain. Accordingly, the adsorption had profound effects on the structure and hydration of the peptide molecules. The high affinity binding to the DMPC water interface decreased the conformational freedom of the polypeptide chains and narrowed the accessible distribution of conformations. The adsorbed peptides sampled mostly turn and bend states, in contrast to the elongated, strand-like conformations found in the aggregates without bilayer.
The strong interactions between peptides and bilayer interface precluded extensive inter-peptide interactions and might explain why adsorption was favored over self-assembly of monomers and small intermediate aggregate species. Furthermore, the molecular kinetics and rates of conformational rearrangement of the peptides at the lipid water interface slowed down, relative to those in water.
All this may cause diverging pathways of peptide aggregation in water and near a phospho-lipid membrane. The observed peptide phospho-lipid interactions should have a concentration-dependent effect on the aggregation mechanism for early and small multimeric aggregates. At large pep-tide concentrations little to no effect is expected, since the spontaneous aggregation does occur in the aqueous phase as reported, even with the membrane interface being saturated by bound peptides. At very low concentrations the adsorption of monomeric or partially aggre-gated species to the interface might slow down the aggregation process, by effectively lowering the peptide concentration in solution even further. The highly favorable binding should hinder conformational rearrangements resulting in extensive peptide-peptide interactions furthermore.
The effective enhancement of peptide concentration at the water lipid interface might play a role for peptide aggregation under intermediary concentrations.
Ultimately, the fine interplay between density (ratio of peptide to lipid molecules), orga-nization and structure of membrane-bound oligomeric aggregates will be crucial for the sub-sequent assembly to amyloid fibrils [13]. Despite initial efforts in the characterization of the lipid-catalyzed amyloid formation [13], further efforts, both from the experimental and the theoretical side are required.
The effect of the lipid head groups and peptide concentration could be further investigated following our presented computational approach. It would be interesting to compare different phospholipid types (e.g. anionic PS lipids) in this context [339]. In order to disentangle the effect of electrostatic interactions completely, it could be also interesting to probe the peptide
aggregation process on a purely hydrophobic surface e.g. near an octane slab [340]. Lastly, the question to what extent the properties of the lipid bilayer itself are altered locally or globally by the absorbed aggregates remains to be studied in more detail. Of particular interest in this regard could be to test if and to what extent conformational transitions in already assembled decamers with highβ-sheet content occur once they interact with a DMPC bilayer surface, and in addition, how this affects the conformational properties of the membrane.
Summary and Conclusions
The cellular environment is a crowded and dense setting in which peptides and proteins are subjected to a complex apparatus and network of regulatory mechanisms. The performed operations include supervision of protein folding, prevention of aggregation, activation of post-translational modifications, assistance in transport to and initiation of degradation. Failures in these mechanisms can result in the formation of a variety of harmful proteinaceous assem-blies, in all likelihood the causative agents of several neurodegenerative disorders subsumed as amyloidosis.
The primary aggregates, such as low-molecular weight oligomers, and the course of events in initial peptide assembly free in solution and on membrane interfaces, however, still lack a detailed characterization. Understanding the molecular basis of nonfibrillar oligomer formation harbors the potential to decipher an important, yet still obscure part of amyloidogenic pep-tide and protein aggregation. The elucidation of the energetic and structural determinants of amyloidogenic aggregation poses an essential challenge to biophysical studies and still needs to rely on the study of simplified model systems. Although it is not straightforward to establish a link between the amyloid phenomena in vivo to in vitro studies, significant advances in the use of model systems have led to the determination of reliable structural models for amyloid fibril formation. Moreover, concepts and insight from theoretical and simulation studies help to describe and understand amyloidogenic aggregation on a molecular level.
In the present thesis, the early stages of peptide oligomerization have been studied by means of MD simulations, as an important complementary tool to experimental studies in order to understand the main forces and conformational transitions which govern oligomeric aggregate formation.
Major findings and conclusions of this thesis are summarized in the following.
6.1 Secondary structure propensities of MD Force Fields
The conformational ensembles obtained with MD simulations of e.g. peptide folding and ag-gregation are inherently dependent on the accuracy of the applied force field. The study of the folding behavior of various peptide sequences in different MD force fields revealed significant and systematic differences in the stability and formation propensity of dominant secondary structure elements. The observations made suggest that in particular the relative stabilities of helical and extended conformations depend on a subtle balance of force field parameters. Indications for different sampling characteristics of the respective force fields, affecting both the kinetics and convergence of the simulations, were also found. It is likely that the discussed deviations in the structural representations are less critical in protein simulations, when studying the native state dynamics.
A particular finding concerns the treatment of electrostatics in biomolecular simulations. It is a common practice nowadays to apply Particle-Mesh Ewald (PME) methods. However, care is required when employing PME in conjunction with force fields and water models which were originally developed using cut-off or reaction-field (OPLS, GROMOS96). In terms of secondary structure propensities the peptides studied revealed a tendency towards sampling β-hairpin structures when employing PME combined with the OPLS and GROMOS96 force fields. The observed similarities between the different force fields support the notion of converging results for biomolecular systems, but remaining differences emphasize the importance of continuous force field development and refinement. Thus a force field bias for folding studies can not be excluded. From the current perspective there is no single best fit solution for peptide folding simulations with today’s non-polarizable force fields, rather a multiple force field or consensus approach is suggested: If computationally feasible to simulate, using more than one suitable force field to address the particular question at hand, and whenever possible, to compare the simulation results to direct experimental data.