Building a new type of protein from scratch: functional 310 assemblies

Helical structures are used extensively in nature to build high-order assemblies and store information. This is largely due to their ability to pack well into highly defined conformational space. Among such helical molecules, the polypeptide alpha helix is the most commonly seen structure, within which 33% of the over 2 million residues are located. It is one of the fundamental constants of biology and widely used as a scaffold in protein design, protein engineering, and biotechnology. Alpha helices are conformationally highly defined with narrow range of backbone torsion and tight helical parameters, as well as the stabilising intrahelical hydrogen-bonding backbone between residues i and i+4. In another word, the alpha helix sits in a deep and narrow free-energy well. This is the reason why alternative structures that exist around the alpha-helical region are less prevalent in nature. They lie on the rim of this well and are therefore thermodynamically less stable.

One of such alternative assemblies is the 310 helices. The name 310 originates from the 3 amino acids per turn (instead of 3.6 observed in alpha) and the 10 atoms within one intrahelical H-bond cycle. It forms a tighter and longer helical structure for the same number of residues in comparison to the alpha helix, with H-bond pattern i to i+3. Although they were in fact discovered earlier than alpha helices, nature seems to have overlooked them when building tertiary or quaternary structure in aqueous media. Therefore, it is of our interest to investigate why nature has not found and exploited 310s evolutionarily and whether they can nonetheless exist or be designed de novo.

Recent work in the Woolfson group has confirmed the solution-state and crystal structure of an octameric 310 helix bundle. Each constituent helix has three types of amino acids with specific roles: (1) Hydrophobic Leu residues that point into the centre of the assembly to achieve helix-helix stabilisation. (2) Electrostatic residue pairs, Glu and Lys, to increase water solubility and further stabilise the assembly through salt bridging. (3) Quaternary amino acid to favour the tighter helical turns. Interestingly, 310-based quaternary structures can form reliably but only when incorporating alpha,alpha-disubstituted amino acids (such as Aib) due to the sharper helical turn introduced by greater steric constraints at the alpha carbon.

Therefore, we are intrigued to diversify the 310-promoting synthetic amino acids palette by incorporating synthetic, quaternary, and polar amino acids into the peptide sequence. Previously, the Clayden group demonstrated an enantioselective alpha-arylation of amino acids strategy via the temporary formation of a second stereogenic centre of imidazolidinyl urea. Such quaternary alpha-arylated amino acids were shown to be inserted without disrupting the helical folding and uses enantiopure amino acid precursors as the source of asymmetry.

310 helix based quaternary structures do not occur naturally in long chains, designing and functionalising them could enable us to explore new chemical space that go beyond what was achieved with traditional alpha helices. Thus, our goal is to explore other sequences using de novo design and incorporate unnatural quaternary amino acids.

1. PEOPLE: We will train students with skills that are in demand across a spectrum of industries from pharma/biotech to materials, as well as in academia, law and publishing. The enhanced experience they receive - through interactive brainstorming, problem and dragons' den type business sessions - will equip them with confidence in their own abilities and fast-track their leadership skills. 100% Employment of students from the previous CDT in Chemical Synthesis is indicative of the high demand for the skills we provide, but as start-ups and SMEs become increasingly important in the healthcare, medicine and energy sectors, training in IP, entrepreneurship and commercialisation will stimulate our students to explore their own ventures. Automation and machine learning are set to transform the workplace in the next 20 years, and our students will be in the vanguard of those primed to make best use of these shifts in work patterns. Our graduates will have an open and entrepreneurial mindset, willing to seek solution to problems that cross disciplines and require non-traditional approaches to scientific challenges.

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