Secondary structure of target β-peptides inside lipid vesicles

As the insertion of the target transmembrane peptides into specific lipid vesicles at definite conditions has already been confirmed by the Trp-fluorescence spectroscopy, the next task was to investigate whether the inserted β-peptides maintained their characteristic helical secondary structures inside the lipidic environment. In analogy to the CD-spectroscopic investigations performed for the peptides into solution (TFE), the same has been carried out to shed light of the secondary structures of those peptides when inside the lipid vesicle.

The peptides, P1 (14-helical D-β³-Val/Ala/Leu triad peptide), P10 (14-helical D-β³-Val-peptide), P11 (12-helical D-β³-Leu-peptide), P2 (10/12-helical D-β³/β²-Val/Ala/Leu-peptide) and P3 (10/12-helical D-β²/β³-Val/Ala/Leu-peptide), were inserted in three different lipid vesicles, DLPC, DMPC and POPC with peptide to lipid (P:L) ratio kept constant at 1:50 along with the peptide concentration at 30 μM. The choice of the lipid vesicles and the peptides were based on the extent of negative hydrophobic mismatch betwwen the peptide and the lipids. The lengths of

all these peptides were comparable while the hydrophobic bilayer thickness of DLPC almost perfectly matched with the hydrophobic core lengths of this peptide and the negative mismatch increased gradually from DMPC to POPC to an extent of 6 Å to 10 Å, respectively. Specific percentages of EtOH was used to compensate the negative hydrophobic mismatch by reducing the lipid bilayer thickness. After the insertion of the peptides into these lipid vesicles at different conditions were confirmed by Trp-fluorescence spectroscopy, the CD-spectroscopic investigations were carried out to check on whether the inserted transmembrane peptides also retained their original secondary helical structures inside lipid environment.

A

B

C

Figure 5.12: CD-spectroscopic profiles of peptides P1, P2, P3, P10 and P11 in DLPC (A), DMPC (B) and POPC (C) large unilamellar vesicles with P:L = 1:50, temperature 50⁰C and Concentration(peptide) = 30 μM

DLPC P1 P2 P3 P10 P11

Maxima 208 nm 201 nm 202 nm 209 nm 206 nm

Minima 193 nm 212 nm 213 nm 192 nm 193 & 218 nm

DMPC P1 P2 P3 P10 P11

Maxima 207 nm 202 nm 201.5 nm 208 nm 207 nm

Minima 192 nm 210 nm 212 nm 191 nm 194 & 219 nm

POPC P1 P2 P3 P10 P11

Maxima 205 nm 201.5 nm 201 nm 206 nm 205 nm

Minima 191 nm 211 nm 213 nm 193 nm 193 & 217 nm

Table 5.3: CD-spectroscopy results of peptides, P1, P2, P3, P10 and P11 in three different - lipids, DLPC, DMPC and POPC at-a-glance

From the results depicted in Table 5.3, it was evident that all the peptides retained their original secondary helical structures inside three different lipids. So, it was confirmed that all the three

three types of synthesized β-peptides with secondary helical structures, 14-helix, 12-helix and alternate 10/12-helix, retained their native secondary helical structures inside lipid bilayer systems. These experiments also showed that when the peptides were inserted inside the lipids with the use of EtOH to compensate the negative hydrophobic mismatch, that did not affect the secondary structures and the native helicities were maintained compared to that in solution (TFE).

Similarly the longer thansmembrane peptides P4 (14-helical β³-Val peptide) and P5 (12-helical β³-Leu peptide), were also tested in three diferent lipid systems, POPC, 22:1(Cis)PC and 24:1(Cis)PC due to the same strategy as described for the peptides P1, P2, P3, P10 and P11 previously. The two peptides were almost of cmparable hydrophobic core length and the bilayer thickness of POPC almost match with the transmembrane core lenghths of P4 and P5. But the negative hydrophobic mismatch gradually increased between P4, P5 and 22:1(Cis) PC and 24:1(Cis) PC from 6 Å to 8-9 Å, respectively. Different percentages of EtOH was used to compensate the negative hydrophobic mismatches by reducing the bilayer thickness of the lipids in order to insert the transmembrane peptides into the lipid membranes.

A

B

C

Figure 5.13: CD-spectroscopic profiles of peptides P4 and P5 in POPC (A), 22:1(Cis)PC (B) and 24:1(Cis)PC (C) large unilamellar vesicles with constant P:L ratio and peptide concentration at 1:50 and 30 μM, respectivel

22:1(Cis)PC P4 P5 24:1(Cis)PC P4 P5

Maxima 207 nm 206 nm Maxima 208 nm 205 nm

Minima 192 nm 190 & 217 nm Minima 195 nm 192 & 219 nm

Table 5.4: CD-spectroscopy results of peptides, P4 and P5 in three different - lipids, POPC, 22:1(Cis) PC and 24:1(Cis) PC at-a-glance

From the CD-spectroscopy profiles showed in Figure 5.13 and the results depicted in Table 5.4, it was confirmed that the longer transmembrane peptides, P4 and P5, retaind their native secondary helical structures inside three different lipid systems, POPC, 22:1(Cis)PC and 24:1(Cis)PC. The results also evidently exhibited that even in case of nagative hydrophobic mismatch, when the peptides were inserted by compensating the mismatch by using EtOH, the native secondary helical structures inside the lipid environment remained the same.

Therefore, to conclude, the native secondary helical structures of the three different types of β-peptides were retained and it was further confirmed by using different lipid systems that resembled from perfect hydrophobic matched sstems to gradual negative hydrophobic mismatch scenario. But in all cases, the secondary helical structures were retained similar to that in solution (TFE).

After the confirmation of the retention of secondary elical structures inside lipid environments, it was further studied whether the stability of the 14-helical and 10/12-helical transmembrane peptides of comparable lengths in a smae lipid differed at different temperatures. In analogy to the stability studies carried out in TFE, the transmembrae peptides, P1 and P2 were selected for the similar stability studies at different temperatures starting from 20⁰C to 80⁰C inside DLPC lipid vesicle. DLPC lipid vesicle was chosen as the length of hydrophobic cores of the peptides, P1 P2, almost perfectly matched with the bilayer thickness of the DLPC large unilamellar vesicle.

POPC P4 P5

Maxima 209 nm 204 nm

Minima 194 nm 191 & 215 nm

A

B

Figure 5.14: A comparison CD-spectroscopic profile of a 14-helical peptide, P1 (A) and a 10/12-helical peptide, P2 (B), at different temperatures in DLPC to determine the peptide stability keeping P:L ration and peptide concentration constant at 1:50 and 30 μM, respectively

It was evident from the Figure 5.14 that even at temperaure as high as 80⁰C, the peptides P1 and P2 retained their native 14- and 10/12-helical secondary helical structures, respectively.

Although the helical contents were reduced gradualy with increasing temperature, but the peptides were stable enough to show their native secondary helical structures even at 80⁰C.

Nevertheless, there was a striking difference in stability when the 10/12-helical peptide, P2, was measured by CD-spectroscopy in solution (TFE) and inside hydrophobic lipid environment (DLPC). The difference was clearly visible by comparing Figure-5.14 (A) and Figure-5.14 (B).

When in solution environment (TFE), the 10/12-helical peptide drastically lost its helical content and stability in the range of 60⁰C-80⁰C. But this destabilizato was missing inside DLPC hydrophobic lipid environment. The 10/12-helical peptide was as stable as the 14-helical peptide when inside the hydrophobic lipid environment (DLPC) at hight temperature (80⁰C).

Therefore, it could be assumed that the lack of helical dipole moment made the 10/12-helical peptide more hydrophobic in nature than that of the 14-helical peptide. So, the hydrophilic environment in solution (TFE) stabilized the 14-helix more than the 10/12-helix and due to this extra stabilization of 14-helix in solution, the stability of 14-helix was more at higher temperature. On the other hand, the environment inside lipid bilayer was hydrophobic which stabilized the more hydrophobic zero-dipole 10/12-helical peptide, P2, more than that of the 14-helical peptide, P1. Due to this extra stabilization of the 10/12-14-helical zero-dipole peptide inside lipid bilayer, it was equally stable as the 14-helical peptide even when lacking the helical dipole moment at high temperatures (80⁰C). Therefore, it was concluded that inside lipid bilayer, the lack of helical dipole moment played a key role in the increased stability of the 10/12-helical intelligent limiting barrier between the intra- and extra cellular environments. This regulation is very important for individual cells to function properly and surviving. There are several therapeutic agents developed by the scientific community that are not permitted by the biological membrane to enter into the cells. [167, 168] So, the therapeutic application of these drugs are predominantly dependent on the development of effective vector agents that are able to get attached to the target drug molecules and deliver them into the cells through the biological