Balance Corporal

Structural Characteristics

Polypeptides may adopt various different types of helical conformations which can be defined and distinguished by geometric properties such as the pitch, radius and handedness of the helix.

Fig. 3.1: Representative structures of the peptoid backbone amide in the (a) trans and (b) cis conformations. The same positioning of the heavy backbone atoms in these two confor- mations applies in peptides. (c) Peptoid backbone dihedral angles, defined as the angles between the 2 intersecting planes defined by 4 consecutive atoms. For example, ω rep- resents the rotation around the C-N bond and is the angle between the C-C-N and the C-N-C planes.

The helical character of a peptide can also be characterised by the dihedral angles, ω, φ and ψ, adopted by the peptide backbone in a particular conformation. The pep- toid/peptide backbone dihedral angles, which are defined as the angles between two planes between 4 consecutive backbone atoms, are shown in Figure 3.1c. The ω angle represents the conformation of the peptide bond, which is both planar and 180° in almost all cases. This is referred to as the trans conformation (Figure 3.1a). In rare cases the peptide bond exists in the cis conformation (Figure 3.1b) with ω = 0°. In peptides φ and ψ may take a

3. Experimental Secondary Structural Characterisation

range of values that correspond to particular secondary structural conformations. These can be visualised through Ramachandran plots, a form of 2-D histogram that shows the conformational energy or probability for each pair of φ/ψ dihedral angles in the peptide backbone [8].

Ramachandran plots are a useful medium for visualising peptoid secondary structural preferences, given their structural similarities to peptides. By convention peptide/protein Ramachandran plots are normally plotted over the range -180° - +180° with φ on the x-axis and ψ on the y-axis. The conventions for peptoid Ramachandran plots are slightly different to account for the potential cis-trans isomerisation of the peptoid bond. As such, they are often plotted in pairs, one for the φ-ψ energies for the cis conformation and another for the trans conformation. These are normally plotted with axes for the angles from 0° - 360°. This results in centering of regions commonly populated by peptoid structures.

Fig. 3.2: Representative structures for helical backbones in the 3-10, α and π configurations, viewed from above, looking down the helical axis and from the side, looking along the helical axis. The characteristic hydrogen bonding networks for each structure are shown in green. Backbone structures were generated using Pro-Builder online platform [9].

The secondary structures found in peptides and proteins occupy certain regions of the Ramachandran Plot corresponding to the backbone dihedral angles associated with them. The α-helix is the most common secondary structure in nature. It is a right handed helix and has 3.6 residues per turn with a translation of 1.5 ˚A per amino acid, resulting in a pitch of 5.4 ˚A. The α-helix is stabilised by a distinctive pattern of hydrogen bonds between the amide hydrogen and the carbonyl group four amino acid residues earlier in

3. Experimental Secondary Structural Characterisation

the sequence. This is denoted as (i+4 −→ i) hydrogen bonding [10]. α-helices can also be identified by their backbone (φ, ψ) dihedral angles, where adjacent φ and ψ angles sum to approximately -105°, with typical helices adopting the angles (φ, ψ) = (-60°, -45°) [11]. α-helices are found in peptides which vary greatly in length, from sequences as short as 4 amino acids [12] up to 200 amino acids and above [13]. This conformation is often adopted by peptides in hydrophobic environments such as the lipid bilayer interior and is rarely stable in aqueous environments due to the relative weakness of the hydrogen bond network, which does not compensate sufficiently for the entropic cost of folding [14–16].

The second most commonly observed peptide helix, the 3-10 helix, is a 3 residue per turn, right handed helix with a pitch of 6 ˚A. These helices have an (i+3 −→ i) hydrogen bonding pattern and exist most commonly as very short sections of the peptide chain (normally a maximum of 4 amino acids). The backbone dihedrals angles typically sum to approximately -75°, though there is considerable variation given that these structures tend to exist only in very short sections. The 3-10 helix is slightly more elongated and tightly wound than the α-helix with a translation of 2 ˚A along the helical axis per amino acid, compared to the α-helix 1.5 ˚A [17,18].

A third right handed helical structure, the π-helix is also found in natural peptides. This a 4.1 residue per turn helix with an (i+5 −→ i) hydrogen bonding pattern and a pitch of 4.7 ˚A, making it considerably more compact than either the α or 3-10 helix [19]. π-helices do not adopt specific dihedral angles and are usually only 7 amino acids in length [20]. In all of the helices discussed so far, the omega backbone dihedral, which describes the geometry of the peptide bond is approximately 180° and thus each amide is fixed in the trans conformation. Visual representations of the 3-10, α- and π-helices are shown in Figure 3.2.

Fig. 3.3: Representative structures for polyproline helices, viewed from above, looking down the helical axis and from the side, looking along the helical axis. Figure adapted with per- mission from [21].

An additional pair of helical conformations exist in peptide sequences containing pro- line residues (Figure 3.3). These are therefore known as polyproline helices. Polyproline I

3. Experimental Secondary Structural Characterisation

(PPI) helices are characterised by dihedral angles of (φ, ψ) = (-75°, 160°) with cis amide bonds. These are right handed helices with approximately 3 residues per turn. Proline is the only natural amino acid which has been observed to adopt this conformation. Polypro- line II (PPII) helices on the other hand have been observed in sequences containing not only proline but other amino acids too. The PPII helix is more compact than PPI and is a 3 residue per turn, left handed structure containing trans amides with average backbone dihedrals (φ, ψ) = (-75°, 145°) [22]. Neither of the PP helices are hydrogen bond stabilised as the amide nitrogen is involved in the unique proline side chain structure and therefore cannot function as the H-bond donor. As a result, polyproline peptides may bear some structural similarities to peptoids.

Experimental Characterisation of Helices

The presence of different helical conformations in proteins and polypeptides can be deter- mined by many different experimental techniques including x-ray crystallography, NMR, Infrared (IR) and CD spectroscopy, among others. Each of these has unique advantages and disadvantages. This chapter concerns the characterisation of helical structures by CD spectroscopy and this will therefore be the focus of review here. As discussed in Chap- ter 2, CD has long been used to determine the relative fractions of different secondary structural motifs present in proteins and polypeptides in solution. This technique is par- ticularly sensitive to, and apt for observing structural changes in biomolecules due to environmental changes. However, the ability to distinguish between the subtly different helical conformations that exist in peptides is still debated.

The presences of α-helices is widely reported to produce distinctive features in a pro- tein or peptide CD spectrum. These arise due to the electron transitions associated with the peptide bond in this particular conformation. Transitions from the amide non-bonding π orbital to the anti-bonding π* orbital result in a strong positive band centred around 190 nm and a negative band centred around 208 nm. An additional negative band oc- curs at wavelengths of around 220 nm due to n-π* transition [23]. The subtle backbone rearrangement from α to 3-10 helix has been said by some to translate into minor differ- ences in CD spectra, though others contest that the two structures are indistinguishable. Toniolo et al. suggested that the intensity of the negative band at approximately 208 nm is enhanced in the 3-10 helix relative to the α-helix and the intensity of the positive band at approximately 195 nm is diminished. The ratio of second to first minimum is therefore different in 3-10 helices than α-helices. The theoretical prediction for the ratio of the intensities of the two spectral minima for a 3-10 helix is 0.4, which is consistent with published experimental CD spectra [24,25]. There is overlap of the allowed regions of the Ramachandran plot for the 3-10 and α-helix so a molecule may transition from one struc- ture to the other without losing the characteristics of a helix at any point [25]. However,

3. Experimental Secondary Structural Characterisation

further studies have failed to confirm that the 3-10 helix may present a characteristic CD signature [26]. Polyproline helices have CD spectra which are quite distinct from those of α-helices, as seen in Figure 2.2 (in Chapter 2), exhibiting a single, intense minimum at wavelengths in the region of 205 nm and a slight positive peak beyond 220 nm.