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Content archived on 2024-05-27

Protein engineering for the study of detoxification enzymes and hub proteins

Objective

Proteins that exhibit broad specificity play important roles in different biological processes. These proteins include enzymes that catalyse the chemical transformation of many different substrates and proteins that bind to multiple protein partners. We propose to develop and apply novel directed evolution and chemical genetic methodologies for the study of proteins that exhibit broad specificity, with focus on cytosolic sulfotransferases (SULTs), which detoxify a broad range of xeno- and endobiotics, and proliferating cellular nuclear antigen (PCNA), which binds to multiple protein partners to play a central role in DNA replication and repair. SULTs belong to a large family of detoxification enzymes that exhibit broad specificity and relatively poor catalytic efficiency. It is not clear how SULTs can detoxify a variety of different compounds and what constitutes the molecular basis for their broad specificity. Application of directed evolution methodologies will allow us to identify and isolate SULT mutants with improved catalytic efficiency and novel specificity. These mutants will be thoroughly characterised by applying a variety of biochemical and structural methodologies to provide new insights into the broad specificity, catalytic activity and biological functions of SULTs. In parallel, we propose to develop and apply directed evolution methodologies for the study of PCNA. PCNA is a homotrimeric hub protein that forms a DNA sliding clamp to mediate DNA replication and repair by recruitment of a variety of essential proteins to the DNA template. Very little is known about how these multiple binding choices are regulated or about the importance of the different PCNA-protein interactions at different stages of replication. We propose to generate PCNA mutants with new binding activity and novel specificity, followed by thorough in-vitro and in-vivo characterisation, to study the roles of PCNA-protein interactions in DNA replication and repair.

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Keywords

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Topic(s)

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Call for proposal

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ERC-2007-StG
See other projects for this call

Funding Scheme

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ERC-SG - ERC Starting Grant

Host institution

BEN-GURION UNIVERSITY OF THE NEGEV
EU contribution
€ 1 000 000,00
Total cost

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No data

Beneficiaries (1)

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