TIMCAT · Novel catalytic function in a TIM-barrel scaffold
FP7 — People (Marie Curie Actions)
- Duration
- 2010-06-01 → 2012-11-30
- EU contribution
- €174,241
- Participants
- 1
- Scheme
- MC-IIF
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Results in brief
Novel catalytic function in a TIM-barrel scaffold
The aim of this project is to examine the role of loops in enzyme evolution and function. Concerns about the comparatively low stability of the originally proposed target enzyme, monoTcTIM, thought to be due to the exposure of hydrophobic surface on monomerisation of the native TcTIM dimer, led to a comprehensive bioinformatic examination of structurally characterised TIM proteins to find potential alternative scaffolds. The search targeted monomeric proteins with an un-elaborated TIM-barrel structure that does not have significant extensions interacting with the C-terminal face of the barrel. From over 1000 TIM-barrel structures in the PDB, only three protein scaffolds were identified which met the defined criteria – these were indoleglycerol phosphate synthase, 4-hydroxy-2-oxoglutarate aldolase and phosphoribosylanthranilate isomerase (PRAI). Of these, PRAI looked to be the best candidate. The gene for PRAI from E. coli was cloned, over-expressed and the protein purified. Unfortunately, the activity assay for this enzyme was found to be unsuitable for screening in microdroplets due to the small signal change on successful reaction, which was further complicated by the intrinsic chemical instability of the enzyme substrate. Consequently, the research focus shifted to examining the role of loops in enzyme function of alternative scaffolds, and in particular on developing new methodologies for manipulating loops in proteins. Concurrently, work on microdroplets has focused on developing their usability and utility for protein engineering studies.
Data: CORDIS, © European Union
Project objective
The TIM-barrel fold is the most widely found in nature, making up about 6% of the structures in the Protein Data Bank, and has been found in nature to catalyse reactions from five of the six EC classes. These facts all indicate the extraordinary capacity of the TIM-barrel as a reaction container, and suggest that it may be a versatile starting scaffold into which new catalytic capability can be engineered. In this research programme we will use directed evolution approaches, including state-of-the-art gene shuffling techniques developed in the Hollfelder lab at the University of Cambridge to tengineer novel catalytic function into a TIM-barrel scaffold.
Original text from CORDIS.
Participants
- THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE · CAMBRIDGECoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union