Design of 10/12-helical β-peptide based artificial transmembrane domains

Since the very beginning, the investigation on the effect of helical macrodipole moment on different biochemical and physico-chemical properties of membrane protein has been the main point of interest for this project. Therefore, to shed light experimentally on this topic, it is necessary to be able to synthesize at least two different varieties of β-peptide based model transmembrane sequences, one of which should possess definitive helical macrodipole moment and the other should have no helical dipole moment. The idea is to perform similar experiments with these two types and if they exhibit considerably different results, it could be possible to relate the experimental differences to the helical macrodipole moment. In this process, the most challenging part is to design and synthesize β-peptide based model transmembrane sequence that has almost no helical macrodipole moment.

Reviewing the different helical secondary structures possible for β-peptides, it is evident that the alternate 10/12-helix has almost no helical macrodipole moment. The backbone of 10/12-helix is intrinsically the most stable secondary helical structure, but in presence of hydrophobic side chains, it does not hold anymore. So, there could be two main possibilities to design a β-peptide based transmembrane peptide that folds into an alternate 10/12 helix.

i) Peptides synthesized by alternating chirality of β²-or β³-amino acids:

ii) Peptide synthesized by alternating β²-and β³-amino acids with uniform chirality:

Figure 3.3: Schematic overview of the possible combinations to synthesize 10/12-helical β-peptides

Since, the α-helical secondary structures are better preserved in homochiral peptides,^[145] so the idea of designing β-peptide based transmembrane peptides by alternating chirality is immediately dropped. Therefore, the only option left is the homochiral alternate β²/β³-peptides.

Therefore, the planned structures of the β-peptide based transmembrane sequences are P2´.

Figure 3.4: Preliminary design of the β-peptide based artificial 10/12-helical transmembrane domain, P2´

However, the synthesis of this sequence was unachieved after several attempts with lot of different methods altering different parameters. So, some serious modifications had to be done to simplify the sequence as much as possible keeping the ultimate purpose in mind. The several modifications are:

i) Shortening the overall sequence length.

P2´

ii) Omitting the polar amino acid residues (Lys-) at both the terminals, as the attachment of β³ -Lysines to the resin (2-chlorotrityl chloride), that was selected after several optimizations, was not successful. So, the end polar residues had to be eliminated from the sequence.

iii) Due to the elimination of polar amino acid residues, the hydrophobic amino acids had to be attached to the resin at the C-terminal. So, now there were two possibilities available, attaching a β³-amino acid to make a β³/β²-peptide or attachment of a β²-amino acid to synthesize a β²/β³ -peptide. Both the peptides should technically exhibit the same 10/12-helical secondary structures, but in order to make sure that the β³/β² and β²/β³ -peptides have no different characteristics and effects on the desired physico-chemical and biochemical properties, both of the two types were designed to be synthesized and tested. Since, the rise/residue value for a 10/12-helical peptide is about 2.1 Å, so the length of the hydrophobic core of these peptides are approximately 18-19 Å.

Therefore, the modified 10/12-helical alternate β³/β² and β²/β³-peptides are P2 and P3.

Figure 3.5: Final sequences of the β-peptide based 10/12-helical artificial transmembrane domains; β³/ β²-peptide (P2) and β²/ β³-peptide (P3)

The main idea behind designing this peptide is to test any effect of helical macrodipole moment on transmembrane domain insertion into lipid membranes. It has already been discussed that when the hydrophobic core of the peptide is considerably shorter than the lipid bilayer thickness, a negative mismatch occurs. The negative mismatch can be compensated a number of

P2

P3

ways, but one of the frequent outcomes of the negative mismatch is the non-insertion of the peptide into the lipid membrane and instead it takes a parallel orientation to the membrane at the membrane-water interface region. In order to compensate the negative mismatch, a probable solution is to reduce the bilayer thickness of the membrane. Moreover, as discussed earlier, short chain alcohols, like ethanol, has a distinctive ability to squeeze the bilayer thickness via keeping the fatty acid chains at an interdigitated state. So, the idea is to choose three different lipids in similar type with bilayer thickness gradually increasing in comparison to the length of the hydrophobic core of the transmembrane peptide. The lipid with thinnest bilayer should almost match with the hydrophobic core of the transmembrane peptide and the rest of the two lipids should have bilayer thickness gradually increasing up to a mismatch of approx. 10 Å. Therefore, minimum concentration of ethanol is used to compensate the negative mismatch between the peptide and the each lipid bilayers to the extent where the peptide is inserted into the membrane. Now, by comparing these results between a transmembrane peptide without macrodipole moment (10/12-helix) and a peptide with overall macrodipole moment (14-helix), if it is observed that to compensate similar negative hydrophobic mismatch (keeping in mind that the hydrophobic core length of both the peptides are the same and the three lipids used are also the same), there is difference in the required minimum concentration of ethanol, then it can easily be implied that due to the helical macrodipole moment either fascillitates or hinders the membrane insertion and spanning of the transmembrane domains (Figure 3.6)

Figure 3.6: Schematic representation of the concept to determine effect of helical macrodipole moment on membrane insertion and spanning of transmembrane domains via compensation of negative hydrophobic mismatch with minimum concentration of ethanol

Besides the study on the effect of helical macrodipole moment of the designed β-peptide based transmembrane domains (P2 and P3) on lipid membrane insertion and spanning, it is also intended to investigate on the cellular uptakes of these types of neutral, hydrophobic short β-peptide sequences. Since, more than 90% of the established cell penetrating β-peptides carry several positive charges and are composed mostly with hydrophilic amino acid residues, like lysines or arginines, it woul dbe interesting to see if completely hydrophobic, chargeless, short β-peptides can penetrate cell membranes and enter the cytoplasm. Further to elucidate if there is any difference in uptake nature of the same cell lines between the β-peptide sequences with and without macrodipole moment. In that case, it would be possible to shed light on whether the macrodipole moment also regulate biochemical phenomena, like, cellular uptakes.

So, in this purpose, a short hydrophobic sequence of alternate β²/β³-peptide is designed that has no lysines or tryptophans in it (P6).

Figure 3.7: 10/12 helical short β-peptide sequence as a candidate for cell penetrating peptide (C.P.P), P6

In order to quantify the cellular uptake by Fluorescence activated cell sorting (FACS) technique and visualize the peptide inside the cell by Laser confocal fluorescence microscopy, a fluorophore, 5(6)-carboxyfluorescein (5(6)-FAM) is attached to the N-terminal of the peptide P6 to give rise the fluorescently labeled derivative P7.

P6

P7

Figure 3.8: FAM-labeled 10/12 helical short β-peptide sequence as a candidate for cell penetrating peptide (C.P.P), P7