Structural characteristics of transmembrane α-helices in membrane spanning and at membrane-water interface region

Membrane-spanning α-helices are composed of a stretch of predominantly hydrophobic amino acid residues, so that the hydrophobic face of these α-helices and the acyl chains of membrane lipids can interact with each other by Van der Waals force of interactions in an energetically efficient manner. The principle of hydrophobic matching, in which the hydrophobic length of a transmembrane protein segment matches the hydrophobic thickness of the lipid bilayer, was introduced by Mouritsen and Bloom ^[123] and is believed to be an important determinant in protein-lipid interactions. The α-helix is favorable because this structure satisfies the hydrogen bonding potential of the polypeptide backbone. An alternate structure that lacks a single hydrogen bond would be less stable by about 5 kcal/mol ^[124], and therefore, a continuous helix without turns is most favorable. Typical transmembrane α-helices are estimated to vary in length from 15 to 28 residues ^[125] and may be tilted with respect to the bilayer normal, as can be deduced from available three-dimensional structures of membrane proteins.

Compared to the hydrophobic core of the membrane, the membrane–water interfacial (phospholipid headgroup) region presents a chemically complex environment, which offers many possibilities for non-covalent interactions with protein side chains ^[126]. Therefore, the interface is thought to play an important role in membrane association of proteins and peptides.

The carbonyl moieties of the lipids, the phospholipid headgroups and water molecules around the lipid head groups present opportunities for dipole–dipole interactions and allow hydrogen bonding with appropriate amino acid side chains. In addition, electrostatic interactions might occur between, for example, positively charged amino acid side chains and negatively charged lipid phosphate groups. Residues that flank the hydrophobic membrane-spanning segments of membrane proteins will interact with the interface on each side of the membrane and thereby may determine the precise interfacial positioning of these segments or influence their orientation in the membrane [125, 127-130]

A remarkable feature that pops up in many transmembrane α-helices is the distribution of aromatic amino acid residues. Not only do membrane proteins have a significantly higher tryptophan content than soluble proteins ^[131], they are found with a high frequency near the ends of transmembrane α-helices, which are expected to be located in the membrane interfacial region. ^[132] Localization of aromatic amino acids in the membrane interface is for instance observed in the photosynthetic reaction center ^[133], the potassium channel KcsA ^[134], bacterial porins ^[135], and in gramicidin A ^[136]. In addition, several studies on smaller membrane-associated peptides have shown that there are indeed specific interactions of aromatic amino acid residues with the membrane interface ^[137-139]. A variety of roles have been proposed for

interfacial aromatic residues, including positioning or anchoring the transmembrane segments in the membrane and stabilization of the helix with respect to the membrane environment ^[139]. Another feature that is common in transmembrane proteins is the occurrence of charged residues such as arginine and lysine next to the transmembrane segments, and therefore acting as ‘flanking residues’. Although the role of these charged residues in membrane proteins as topological determinants is well-established ^[140], they may also play a role in membrane anchoring in the interfacial region ^[139].

So, based on these investigations, the peptide sequence of Gramicidin A, a polypeptide antibiotic that forms single ion monovalent cation channels in biological membranes, was chosen as a model to design β-peptide based artificial transmembrane domain systems. The linear gramicidin A is a prototypical channel former that have been extensively used to study organization, dynamics and function of membrane-spanning channels. [141, 142] It is a linear pentadecapeptide antibiotic with a molecular weight of ∼1900. Gramicidin A is produced by the soil bacterium Bacillus brevis, and uniquely consists of alternating L- and D-amino acids. ^[143]

They form well-defined cation-selective ion channels in model membranes ^[144] with conductance of the order of ∼107 ions per second. Due to their small size, ready availability and the relative ease with which chemical modifications can be performed, Gramicidin A serves as an excellent model for transmembrane channels. Therefore, it has been a logical approach to consider the linear hydrophobic peptide sequence of Gramicidin A as the basis for designing β-peptide based synthetic transmembrane domain systems in order to address the shortcomings of α-peptide based transmembrane domain systems via peptidomimetic strategy.

Figure 3.1: Peptide sequence of Gramicidin A

So, the apolar amino acid triad, Ala/Leu/Val, is chosen as the hydrophobic core sequence for the model β-peptide based synthetic transmembrane domain system. A tryptophan is thought be incorporated towards the C-terminal of the sequence mainly to verify the location of the transmembrane sequence in solution or in lipid bilayer by detecting the tryptophan

fluorescence. It is also planned to add at least one lysines on each C- and N-terminals of the sequence, so that the polar residues could favourably interact with polar lipid headgroups in the membrane-water interface region. Since, the effect of the tryptophan towards anchoring the peptide sequence to the membrane is still debated, so addition of extra tryptophans for this purpose is initially excluded to make the design as simple as possible keeping extensive synthetic challenges in mind.

Although the L-enantiomers of amino acids are mostly found in nature and in natural proteins are composed of L-amino acids. Nevertheless, repeated studies have exhibited that the unnatural D-amino acid counterparts might have serious advantages in synthetic peptide design. For example, proteins or peptides composed of D-amino acids are often observed to have higher enzymatic stability and are less prone to enzymatic degradation in presence different proteases.

That is exactly what one of the most important reasons for choosing β-peptides to design synthetic transmembrane domains as β-peptides are also reported to exhibit stability in presence of protease enzymes. Therefore, the idea is if D-enantiomers of β-amino acids are used to design the β-peptide-based transmembrane domains that might prove to be extremely advantageous towards added enzymatic stability. So, D-enantiomers of the selected β-amino acids are used to access the designed transmembrane domain system. On the other hand, it has also been observed that D-amino acids have the ability to form more stable secondary structures with less number of residues. Hence, this also perfectly coincides with the reason why the β-amino acids are chosen instead of the α-β-amino acid counterparts.

It has been reported that alpha helices are better preserved in homochiral, rather than heterochiral, peptide sequences. ^[145] Therefore, the alternating D/L chirality of Gramicidin A is dropped and instead a homochiral D-amino acid repetitions are considered for the design of β-peptide based artificial transmembrane domain systems. The sequence length is also kept short between 10-12 amino acid. There are two main reasons behind keeping shorter sequence;

i) The shorter the sequence length of the β-peptides with helical dipole moment less is the magnitude of their helical macrodipole moment as the overall macrodipole moment is the sum of the individual dipole moments of the amino acids. Therefore, it would explain whether minimal difference in the value of helical dipole moment could exhibit any significant change in the physico-chemical or biochemical behaviors of the transmembrane β-peptides.

Ii) Evident from the previous literature, there is considerable challenges to synthesize such types of hydrophobic β-peptides. Therefore, keeping it as short as possible would increase the chance of getting successful.

Hence, the general structure of the designed β-peptide based artificial transmembrane sequence is depicted in Figure 3.2.

Figure 3.2: Schematic representation of general structure of a designed β-peptide based artificial transmembrane sequence