(S)-3-O-Geranylgeranylglyceryl Phosphate Synthase (GGGPS) is a TIM (βα)8 barrel protein found in Archaea and the enzyme catalyzing the first step in the biosynthesis of archaeal membrane lipids. The TIM (βα)8 barrel protein fold is thought to have evolved by duplication and fusion of (βα)4 half barrels. We propose that the GGGPS has also evolved from (βα)4 half barrels. One way to test this hypothesis is to generate putative half-barrels experimentally. GGGPS from Archaeaglobus fulgidus, is a dimer of (βα)8 barrels. Thus, before constructing half barrels, a stable monomer is needed to be engineered.
Introducing three substitutions into the dimer interface formed the GGGPS monomer. AUC showed ~50 % of the protein is in the monomeric state. CD experiments confirmed that the engineered protein was properly folded but had decreased thermal stability. In an enzymatic assay, the monomeric GGGPS protein proved as active as the WT protein on a subunit basis.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/29571 |
| Date | 25 August 2011 |
| Creators | Kharbanda, Neha |
| Contributors | Pai, Emil F. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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