Influence of Peptide Sequence

The results obtained from DSC and FT-IR experiments for DPPG-peptide mixtures are compared to each other with respect to the different peptide sequences. Based on the shift of the transition enthalpy ΔH, the transition temperature Tm and the peptide secondary structure, the peptides are grouped into three main categories (see Table 3.5.1).

Table 3.5.1: Comparison of the effects observed after binding of different proteins[198] or our model peptides to DPPG bilayers. Their effect on the transition enthalpy ΔH and the transition temperature Tm of the lipid component is denoted with arrows, where ↑ means an increase, ↓ a decrease and → no differences compared to pure lipid.

protein [198] mode of action ΔH Tm peptide structure

literature

ribonuclease electrostatic binding ↑ → polylysine electrostatic binding ↑ ↑ cytochrome c fluidization of bilayer

+ hydrophobic interaction

↓ ↓ gramicidin A non-polar interactions ↓ →

own results

1a (K)5, (KAA)4K electrostatic binding ↑ ↑ random 1b (KG)4K, (KGG)4K electrostatic binding ↑ ↑ random and turn 2a (KA)4K, (KAbu)4K electrostatic binding

+ β-sheet formation

↓↓ ↑↑ antiparallel β-sheet to random transition 2b (KV)4K, (KL)4K electrostatic binding

+ β-sheet formation

↓↓ ↑↑ stable

antiparallel β-sheet 3 (RG)₄R, (RA)₄R electrostatic binding

+ hydrophobic interaction no/weak binding?

↓ → random

DPPG mixtures with the peptides (K)₅, (KG)₄K, (KGG)₄K and (KAA)₄K belong to the first category, 1a or 1b, where at a charge ratio of 1:1 the enthalpy and temperature of the lipid phase transition are increased after peptide binding. This is the case when electrostatic effects are dominating, i.e. the screening of charges of the lipid headgroup is the main effect of binding, resulting in a stabilization of the gel phase. For a complete shielding of all headgroup charges an increase in T_m of 5.5 K would be expected [225]. This was found for binding of oligolysines or polylysines to negatively charged membranes [104, 198]. Shortening of the lysine side chain leads to a larger increase in Tm as theoretically predicted [123]. The upshift of Tm of up to 4 K for the peptides in category 1 is slightly below the theoretically predicted increase because of additional hydrophobic interactions of the lysine side chain.

Summary of Bilayer Experiments

The two subgroups 1a and 1b are due to the different structures of the bound peptide strands.

In both categories, the added peptide adopts an unordered structure when bound to DPPG bilayers. Additionally, in the subcategory 1b the peptide backbone of (KG)₄K and (KGG)₄K seems to be flexible enough due to insertion of glycine spacers to adopt an unordered structure with a high content of β-turns. It could be concluded that these peptides bind mainly due to the electrostatic interaction to the lipid bilayer surface without any incorporation into the hydrocarbon region.

The second class of peptides are the more hydrophobic ones with the general structure (KX)4K with X = A, Abu, V, and L. After binding they lead to a much larger increase of the transition temperature combined with a decrease of the transition enthalpy. An increase of the phase transition temperature by 19 up to 23 K is far above the value predicted for purely electrostatic binding, thus an additional stabilizing effect must be present. For binding of the peptides (KX)4K, the formation of β-sheet structures is an additional driving force. The possible arrangements of the peptides and a graphical representation are given in section 3.5.3. Results from FT-IR measurements show that this class can also be subdivided into two subclasses determined by the stability of the secondary structure of bound peptide.

In subclass 2a the peptides (KA)4K and (KAbu)4K with intermediate hydrophobicity form intermolecular β-sheets upon binding to gel phase bilayers, which convert to an unordered structure at the temperature of the lipid chain melting. Thus, the lipid chain melting is coupled to the structural transition of the peptide. This additional level of self-organization of peptides bound to the lipid bilayer stabilizes the gel phase of the lipids, whereas the unstructured peptides of category 1 only show minimal stabilization. The defined structure of the adsorbed peptide leads to a distinct separation of the lipid bilayer from the peptide layer without any incorporation into the acyl chain region. Vesicle aggregation, as observed by visual inspection and by cryo-TEM, is caused by a cross-linking of the bilayers via the peptide and a dehydration of the bilayer surface after peptide binding. This is clearly evident from the change in the C=O vibrational band of the lipids. When the peptide undergoes the transition into an unordered structure, this dehydration is reduced and the C=O groups become more hydrated again to an extent very similar to the hydration level of pure DPPG in the fluid phase.

The class 2b is composed of the more hydrophobic peptides: (KV)4K and (KL)4K. Their influence on the thermotropic behavior of the DPPG system is similar to the group 2a, as they also form intermolecular β-sheets after binding to gel phase DPPG and increase the transition

temperature much more than expected from simple charge screening effects. The important difference is the stability of the bound intermolecular β-sheet due to the increased hydrophobicity (see section 3.5.3). The antiparallel β-sheet on top of the lipid bilayer remains also stable when the lipid performes the phase transition from gel into the liquid-crystalline phase. A penetration of bulk water into the lipid headgroup region is hindered, so that the headgroup region stays less hydrated even in the liquid-crystalline phase. For (KL)₄K, the transition temperature decreases to some extent compared to (KV)4K. This indicates that the most hydrophobic amino acid used in this study seems to slightly perturb the lipid bilayer, meaning that the hydrophobic side chain might be in contact with the lipid headgroup region.

The third group of peptides are the arginine containing peptides (RG)4R and (RA)4R which behave differently than those mentioned before. The transition enthalpy is slightly lowered at a constant transition temperature. Similar effects for binding to DPPG vesicles were observed for peptides with non-polar interactions without any electrostatic contributions, e.g.

gramicidin A [198]. However, the arginine plays an important role in these interactions, showing a more complex behavior. The superposition of the counteracting effects, namely the binding to the bilayer upon electrostatic interaction, the formation of bidentate hydrogen bonding, and the hydrophobic interaction of the guanidinium moiety with the lipid headgroup, causes a zero net effect on the phase transition.

Figure 3.5.1: Binding of the peptides (KX)4K to DPPG membranes studied by DSC and FT-IR: The hydrophobicity of the uncharged amino acid X determines the secondary structure of the bound peptide. For hydrophilic G unordered conformations are detected. For X = A or Abu, the secondary structure changes with temperature, whereas for X = L or V, stable β-sheets are formed at all temperatures. The bound β-sheets strongly increase the phase transition temperature of DPPG.

Summary of Bilayer Experiments

Comparing the results shown in Table 3.5.1 with the matrix of the peptides in Figure 2.3.1 reveals a distinct influence of the peptide sequence on the termotropic behavior of DPPG bilayers after peptide binding. The effects upon peptide binding on the phase behavior of DPPG are larger comparing peptides of two different groups as the difference within the groups themselves. The introduction of an uncharged amino acid reduces the hydrophobic contribution of the lysine side chain and increases the electrostatic effect of charge screening.

Tuning the hydrophobicity of the side chain of the uncharged amino acid enables the formation of an additional level of self-organization resulting in an extensive stabilization of the lipid gel phase. Exchanging lysine by arginine further enhances the hydrophobic contributions of the side chain counteracting the electrostatic effect.