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

Informazioni relative al progetto

PyroPhosphoProtein

ID dell’accordo di sovvenzione: 700124

DOI

10.3030/700124

Progetto chiuso

Data della firma CE 8 Marzo 2016

Data di avvio 1 Aprile 2016

Data di completamento 31 Marzo 2018

Finanziato da

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Costo totale

€ 183 454,80

Contributo UE

€ 183 454,80

Coordinato da

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.)

Periodo di rendicontazione: 2016-04-01 al 2018-03-31

Sintesi del contesto e degli obiettivi generali del progetto

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.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

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.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

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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