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| Author(s): |
Paulo Henrique Conaggin Godoi
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Química de São Carlos (IQSC/BT) |
| Defense date: | 1998-04-29 |
| Examining board members: |
Glaucius Oliva;
Maria Teresa do Prado Gambardella;
Carlos Frederico Martins Menck
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| Advisor: | Glaucius Oliva |
| Abstract | |
ln this project it is intended to solve the three-dimen sional structure of a recently identified protein, Thi1, possibly related to DNA repair in Arabidopsis thaliana and which has also be shown to act in the biosynthesis of the thiazole ring of thiamin (vitamin B1) in yeast. The catalytic mechanism for this protein might be also proposed if possible. The cloned thil gene and its deleted form, lacking the bases coding for the first 55 amino acids, previously identified as a transit peptide for the chloroplast, were subcloned into a pET-28 plasrnid vector and inserted after a 6xHis tag sequence. This procedure allowed large scale expression of both proteins however only the deleted protein form could be purified on an immobilized metal chelate affinity column. The recombinant protein was crystallized at 4°C as thin plates in two different conditions, on which the best results were seen in 100mM Bicine pH8.8 containing 800mM (NH4)2SO4. Even then new experirnents airning at improving in the crystal habit are in progress. Data collection is intended to be performed on a home source and in a syncrotron facility. Due to lack of homology with any other protein of known three-dirnensional structure, the approach to be adopted is that of multiple isomorphous replacement requiring at least two different heavy atom derivatives of the protein. (AU) | |
| FAPESP's process: | 95/06925-6 - Crystallographic studies of SOR-1 protein, an Arabidopsis thaliana gene product involved in DNA repair of oxidative lesions |
| Grantee: | Paulo Henrique Conaggin Godoi |
| Support Opportunities: | Scholarships in Brazil - Master |