Martin, Freddie

English

Protein design requires an understanding of sequence-to-structure relationships. While knowledge in the field continues to grow and computational methods become increasingly powerful, there are areas lacking understanding of how an amino acid influences the final protein fold. Using a rational and systematic experimental design allows a logical study of the protein’s properties and function. α-helical coiled coils (CCs) are one of the best-understood protein folds, comprised of two or more α-helices assembling with interdigitating amino acids. Through empirical, rational, and computational design, the CC landscape has been explored, discovering structures never observed in nature and creating functional proteins, from first principles. Yet, our understanding of coiled-coil dynamics and sequence-to-structure relationships is incomplete.Chapter 2 of this thesis explores the dynamics and kinetics of a set of designed CCs, ranging in oligomeric states from dimers to heptamers. A fluorescence-based assay developed herein analyses the orthogonality of these homo-oligomeric CCs and monitors the rates of exchange. The data reveal that all basis-set peptides interact with each other; thus, they are not orthogonal. This is apart from a dimeric CC which contains a buried asparagine residue at its hydrophobic interface. Type-1 CCs and type-2 CCs were found to exchange promiscuously with themselves but not with each other. We successfully characterise a new orthogonal trimer based on recent tetramer designs, using glutamine residues at the g and e positions of the coiled-coil heptad. Further, a new set of orthogonal type-2 pentameric CCs are designed with buried glutamine residues. However, this mutation disrupts the assembly of the type-2 CC and does not offer the same orthogonality observed in the dimeric and redesigned type-1 CCs.Chapters 3, 4, and 5 explore the g and e positions of the type-2 CC interface, using a series of rational, functional, and quaternary amino acids to understand their role in determining the CC structure. First, a systematic study of the type-2 CC sequence g and e positions is described, using glycine, alanine, serine, and threonine. Type-2 CCs, with the favourable amino acids at the core a and d positions, form α-helical barrels. These are CCs with five or more helices and a central channel to which small molecules can bind. Initially, the g and e positions are considered in isolation from one another, and then further variants with systematic mutations at both g and e are studied. All the peptides are characterised in solution and crystal states. While the g-position variants all behave as α-helical barrels and show a clear pattern in their sequence-to-structure relationship and the packing of the helices, e does not. Instead collapsed α-helical bundles and megadalton- fibral assemblies are observed. The e position does not contribute the same rational control of the structure as the g position. Lastly, variants with mutations g and e form β- amyloids, α-helical barrels with reduced stability, or are unfolded.Next, using the knowledge gained, histidine is studied at the g and e positions to design a series of pH stimulus-responsive α-helical barrels. In solution, at neutral pH, peptides with histidine at g or e behave as α-helical barrels, whereas the double histidine variant does not. When pH is reduced, the peptides are no longer able to bind to small molecules. In the crystal state, all peptides form tetrameric CCs. Quantification of the switch and analyses of interactions in structures find that, due to the different interactions at the g and e positions, the pKa is significantly shifted.Lastly, we explore the use of the quaternary amino acid, α-aminoisobutyric acid (Aib). Mutated sequences of peptides with alanine at the b and c positions in type-1 CCs and the g and e positions of type-2 CCs are characterised. In type-1 CCs Aib does not affect the stability of the assembly; however, the oligomeric state is reduced. In type-2 CCs, collapsed assemblies with mixed parallel-antiparallel helix orientations are observed. To rationalise the change in the assembly structure, we analyse the phi and psi of the structures, steric clashes, and area of exposed hydrophobic residues. While significant steric clashes and shifts in the phi and psi are observed, neither can explain the change in structure. The change in structures is found to be due to increased hydrophobicity of the side chain in Aib

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Exploring the dynamic and conformational landscape of α-helical peptide assemblies

https://research-information.bris.ac.uk/files/348516934/Final_Copy_2022_12_06_Martin_F_PhD_Redacted.pdf

Exploring the dynamic and conformational landscape of α-helical peptide assemblies

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