De novo design of an UpConverting metalloProtein

Project Information

UCLnProt

Grant agreement ID: 841956

DOI

10.3030/841956

Project closed

EC signature date 11 April 2019

Start date 2 September 2019

End date 1 September 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

THE UNIVERSITY OF BIRMINGHAM
United Kingdom

Periodic Reporting for period 1 - UCLnProt (De novo design of an UpConverting metalloProtein)

Reporting period: 2019-09-02 to 2021-09-01

Summary of the context and overall objectives of the project

During this fellowship a new class of Lanthanide coiled coils (LCC) were prepared, designed de novo (“from scratch”), to generate bi- and tri- homo- and hetero- metallic derivatives which are capable of binding the full range of Ln(III) ions. These LCCs combine the attractive photophysical properties of Ln complexes, with de novo designed coiled coil scaffolds capable of selectively binding different Ln ions at well-defined locations and tuneable distances.
This project developed a library of coiled coils, a new class of ligands for lanthanide ions, and generated a series of lanthanide complexes. The traditional field of lanthanide chemistry has focussed primarily on the use of traditional small molecule ligands. This work therefore provides a new class of ligands to the lanthanide coordination chemistry toolbox. Furthermore, lanthanide biochemistry is a field only a decade in the making. Consequently, the coordination of lanthanides to miniature protein scaffolds, our coiled coils, and the interrogation of resulting coordination chemistry, provides useful insight into lanthanide biochemistry.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The work of this 2-years fellowship involved the design, synthesis on an automated microwave assisted peptide synthesiser using standard Fmoc-amino acid solid-phase peptide synthesis (SPPS), purification by RP-HPLC and characterisation by ESI-MS, of a library of peptides designed to fold into coiled coil (CC) with binding sites suitable for the coordination of one, two or three Ln(III) ions at a well-defined, controllable, and therefore tuneable, distances from one another. Lanthanide coordination was interrogated using a range of spectroscopic techniques, including UV-visible, fluorescence and circular dichroism spectroscopy. Attempts were also made to obtain structural information through single crystal x-ray diffraction, yielding one crystal structure suitable for publication.
Uni-, bi- and tri-metallic coiled coils were designed and interrogated. Comparisons between the different designs and the spectroscopic outputs led to the establishment of some key structure-function relationships, which ultimately allowed for the more precise tuneable design of “functional” lanthanide sites.
The results obtained have been communicated to the international scientific academic community in several national and international conferences. In total this results in 8 posters and 1 oral communication over the last two years, in addition to weekly and monthly presentations at regular group meetings. Despite the COVID-19 lockdown and subsequent restrictions, I was also able to deliver several talks on the project to the general public (within the UK). Pleasingly these activities are still ongoing and continue despite the project formally coming to an end.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In the last decade interest in lanthanide biochemistry has greatly increased, since it was discovered that lanthanides are biologically relevant. Furthermore, lanthanides exhibit interesting photophysical properties exploited in many useful applications at the interface of biology and inorganic chemistry. The lanthanides are used routinely in technology widely used throughout our daily lives. Therefore the greater understanding of lanthanides coordinated to a new class of ligands offers opportunities across a wide range of industries (e.g. catalysis, waste recovery and recycling, therapeutics, sensing to name but a few). Coupled with the insight that these model protein ligands can provide into enhancing our current understanding of native lanthanide biochemistry more widely.
De novo designed peptides allows one to effectively engineer 3D metal-binding sites within a peptide scaffold with predictable secondary, tertiary and quaternary structure, for metal ion coordination. This offers the synthetic advantages of small molecule complexes, whilst retaining the core elements, and importantly advantages, of natural proteins. De novo designed peptides offer a simplified and robust scaffold with which one can more readily establish important structure–function relationships. As for other metals, the coordination environment plays an essential role in the coordination chemistry and resulting lanthanide complexes physical properties. We developed several designs able to bind two or three lanthanide ions and obtain both homo- and hetero- bimetallic coiled coils. The stability and the photophysicial properties of these scaffolds were evaluated and on the base of these results new improved sequences were redesign and synthetized.

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