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Experimental study of the relevance of the structural network hubs on the catalytic and structural properties of a beta-glucosidase

Grant number: 11/18266-1
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): January 01, 2012
Effective date (End): July 31, 2016
Field of knowledge:Biological Sciences - Biochemistry - Enzymology
Principal Investigator:Sandro Roberto Marana
Grantee:Valquiria Pianheri Souza
Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:08/55914-9 - Development of beta-glycosidases designed to improve the efficiency of noncomplexed cellulase systems, AP.BIOEN.TEM

Abstract

The protein structure can be represented as a network where amino acids are the nodes and their interactions are edges or connections. The structural protein networks are classified as "small world" and "scale free" indicating that amino acids are connected in groups and these groups are interconnected through a few highly connected nodes. The presence of these central nodes, called hubs, contributes to determining shorter routes (sets of connections) between different nodes on the network. Because of these properties, it was suggested that the structural protein network is "robust", that is, random mutations tend to reach the nodes with low degree of connection and therefore cause only small local effect and do not propagate damaging the overall structure of the protein. On the other hand, mutations directed to hubs or its proximity can spread and significantly modify the protein structure. However, despite "robustness" is observed for other network systems, it has not yet been experimentally demonstrated to structural protein networks. The goal of this project is to test this hypothesis, that is, to test experimentally the significance on the propagation of mutational effects of amino acids that are the hubs of the structural network of the beta-glucosidase Sfbgli. To do this, a set of amino acids that interact directly with a hub of the Sfbgli structural network and a set of amino acids that interact directly with a non-hub residue will be replaced by site-directed mutagenesis. Following the effects of these mutations on parameters related to enzymatic activity, substrate specificity and the structural stability of this beta-glucosidase will be assessed by enzyme kinetics and biophysics experiments. (AU)