Site-selective chemical pyrophosphorylation of proteins using tag-and-modify approach.

Informations projet

PyroPhosphoProtein

N° de convention de subvention: 700124

DOI

10.3030/700124

Projet clôturé

Date de signature de la CE 8 Mars 2016

Date de début 1 Avril 2016

Date de fin 31 Mars 2018

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 183 454,80

Contribution de l’UE

€ 183 454,80

Coordonné par

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
United Kingdom

Periodic Reporting for period 1 - PyroPhosphoProtein (Site-selective chemical pyrophosphorylation of proteins using tag-and-modify approach.)

Période du rapport: 2016-04-01 au 2018-03-31

Résumé du contexte et des objectifs généraux du projet

Site-selective protein modification is a powerful chemical biology tool that allows protein labelling, tracking in cells, studying enzyme functions, or creating better therapeutics. Different methods were developed for construction of precisely modified proteins. One of the powerful strategies is a two-step “tag-and-modify” approach, which takes advantage of unique chemical properties of dehydroalanine (Dha). In the first step, Dha (“tag”) is introduced into the position of interest providing protein with a reactive handle that is further modified. The approach is particularly useful for installation of various post-translational modifications (PTM) and natural amino acid mimics since it leaves ‘zero scar’ in protein structure. This project further broadened the scope of “tag-and-modify” approach by developing methods for (a) direct protein pyrophosphorylation, (b) installing histidine isosteres into enzyme active sites using unprecedented aza Michael addition to Dha. Moreover, stereoselectivity of thia-Michael addition to Dha in protein was explored.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

The results generated during the project have contributed to state-of-the-art by broadening a set of tools available for protein modification using unique chemical properties of Dha. The method for site-selective chemical pyrophosphorylation of full length protein giving ppCys (mimic of natural ppSer) and its non-hydrolysable analogue was developed. Next, an unprecedented strategy for post-translational creation of β,γ-C,N bonds in proteins using Dha chemistry (aza-Michael addition) was described. This approach was further used to introduce histidine regioisomers, Hiso and azaHiso, into enzyme active sites for precise probing contributing roles of histidine size and H-bonding toward enzyme activity. Finally, stereoselectivity of thia Michael addition to Dha in protein was investigated. The results of the project resulted in scientific publication in ACS Central Science journal (ACS Cent. Sci. 2017, 3, 1168), review on protein modification via dehydroalanine chemistry (Curr. Opin. Chem. Biol. 2018, in press) and were presented and discussed at international conferences.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

Protein modification via Dha allows ‘zero scar’ protein mutagenesis that in turn allows creation of precise biomimics of natural amino acids and/or post-translational modifications. During the project, a review was published discussing approaches to modify proteins via dehydroalanine with emphasis on recent applications ranging from creating modified nucleosomes to modulating protein functions. This review is expected to be useful source of information for the chemical biology community.
Until now, many proteins have been found to be pyrophosphorylated in vitro but the real significance of protein pyrophosphorylation in vivo remains poorly understood due to lack of chemical biology tools to study this novel post-translational modification. Here we developed a novel method for preparation of pure, well defined pyrophosphorylated proteins and their stable analogs offering long awaited tool for probing of pyrophosphorylation function.
Previously, β,γ-C,S (thia-Michael addition); β,γ-C,Se (selena-Michael addition) and β,γ-C,C (radical addition) bonds were formed post-translationally through Dha modification in proteins. Here, β,γ-C,N (aza-Michael addition) was described providing a novel protein conjugation method as well as a general tool for installing histidine mimics into proteins. These mimics were shown to be useful in dissecting histidine contribution to enzyme activity.
The project complied with the basic principles of the European Research Area by fostering research training, collaboration, mobility and knowledge transfer. It has enabled scientific and personal development of individuals and further promoted Europe as centre of research excellence.

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