Abstract
Oligomers of the achiral amino acid aminoisobutyric acid (Aib) make rigid 310 helices. Thispossesses a well-defined linear secondary structure but is rare in proteins, but often found in
membrane-active antibacterial peptides. 310 helices formed from aminoisobutyric acid (Aib)
readily insert into membranes and, once embedded, have been shown to discharge a pH
gradient across a membrane with activity dependant on foldamer length and terminal
functionality. The sterically hindered nature of Aib makes the synthesis of membrane length
oligomers a laborious process.
This thesis describes the successful development of solid-phase peptide synthesis methodology
to facilitate Aib-on-Aib couplings over 100 times faster than by solution-phase methods. This
methodology has been exploited in coupling up to 17 consecutive Aib residues on a solid
support.
These long Aib-oligomers were also studied for the first time in water, made possible by
inclusion of amino acids favouring aqueous solubility and 310-helicity was observed in water
but only in sequences containing >14 consecutive Aib residues.
This methodology has also allowed the freedom to incorporate Aib and other quaternary amino
acids into peptide sequences. Chapters 4 and 5 describe the inclusion of Aib and quaternary a
-arylated amino acids respectively into known a-helical coiled coil systems. Chapter 4
describes the mutation of alanine with Aib in a selection of a-helical coiled coils. This resulted
in a change in oligomeric state of coiled coils where a parent tetramer is reduced in size to a
trimer upon incorporation of Aib. The presence of Aib in larger barrels also caused disruption
where both symmetry and oligomeric state were disrupted with even small quantities of Aib
present.
Chapter 5 describes a simple study to observe the tolerance of quaternary a-arylated amino
acids in a-helical systems. Both enantiomers of an arylated analogue of alanine were studied
in a nascent system and a well folded peptide system and both were well tolerated in each.
Although it was a minimal difference, the quaternary residue based on D-alanine was slightly
more preferred than that of L-alanine, essentially meaning that the quaternary residue with the
larger side chain facing the opposite direction to those of L-amino acids is more tolerated in
peptide sequences made of those L-amino acids.
Finally, chapters 6 and 7 use this methodology in the development of 310 helices with
transmembrane properties. Instead of simply synthesising Aib oligomers, sequences were
designed to be water-soluble and to promote association in the membrane phase. Chapter 6
describes sequence design from first principles where charged residues were used to encourage
the formation of amphipathic helices. The desired 310-helical geometry was not observed in
solution, but some peptides did show proficiency in discharging a pH gradient across a
membrane.
Chapter 7 describes a different approach to design amphipathic 310 helices, derived from de
novo a-helical coiled coils. With this approach, 310-helicity was observed in solution, but it
was dependent of the length of the peptide. Only the longer peptides of lengths 24 residues or
greater showed 310-helicity whereas shorter peptides adopted a more a-helical secondary
structure. Interestingly, the longer, 310-helical peptides were quite poor at discharging a pH
gradient across a membrane but the shorter sequences, still of transmembrane length showed exceptional ionophoric activity in a membrane, even surpassing that of the antimicrobial
peptide alamethicin.
| Date of Award | 25 Jan 2022 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Jonathan Clayden (Supervisor) & Dek N Woolfson (Supervisor) |