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Multidisciplinary approach to the analysis of enzyme catalysis, protein stability and folding

Objectif

In this multidisciplinary cooperation the methods of molecular modelling, structure determination and microbial techniques are applied to the analysis of protein stability, folding and functional properties, with the aim of understanding the underlying mechanism at the atomic level.
The research associated with this project utilizes prior work associated with the protein engineering of Barnase which has generated some 50 mutants all in relation to stability and activity.

The results which have been obtained to date can be divided into 3 categories:
development of research tools;
results aiming at the understanding of the mechanism of folding and stability;
results aiming at the understanding of the mechanism of enzyme kinetics.

Development of the research tool box:
In addition to various X-ray crystal structures which are already available for Barnase solution structure of Barnase has been determined by nuclear magnetic resonance (NMR).

Resultsaiming at the understanding of the mechanism of folding and stability:
The thermal stability of wild type Barnase (WT) and the T16S-mutant was investigating using high-sensitivity Differential Scanning Calorimetry (DSC).

Results aiming at the understanding of the mechanism of enzyme kinetics:
These include a study on the subsite specificity of the transesterification reaction catalysed by Barnase; modelling and analysis of the Barnase utilizing a pentanucleotide transition state model; the molecular dynamic simulation of barnase in the presence of 5 phosphate ions CH3OPO4 and the simulation of histidine protonation and deprotonation.
In the present project a multi-disciplinary approach is applied to the analysis of protein stability, folding and functional properties, with the aim of providing understanding at the atomic level of the underlying mechanisms.
It integrates experimental studies including site directed
mutagenesis, denaturation studies, analysis of catalytic parameters, as well as structural studies by x-ray diffraction and 2D NMR techniques, with theoretical analyses that combine molecular mechanics, and knowledge based modelling with dynamical simulations and free energy calculations. It will moreover be applied to a unique system, the enzyme barnase, and related microbial endonucleases which has proven to be a superior paradigm for such studies. This system can also be used to analyze protein-nucleic acid interactions and to experiment with design of G-binding peptides.

Thème(s)

Data not available

Appel à propositions

Data not available

Régime de financement

CSC - Cost-sharing contracts

Coordinateur

Gist - Brocades BV
Contribution de l’UE
Aucune donnée
Adresse
1,Wateringsweg
2611 XT Delft
Pays-Bas

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Coût total
Aucune donnée

Participants (3)