Structural studies of glycoside hydrolyses in solution using small-angle scattering (SAS) techniques

• Grant number: 14/00769-5
• Support type: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): June 01, 2014
• Effective date (End): March 31, 2019
• Field of knowledge: Biological Sciences - Biophysics
• Principal Investigator: Igor Polikarpov
• Grantee: Vasilii Piiadov
• Home Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

The Glycoside Hydrolases (GHs) play a key role in a number of biomedical processes and industrial applications. Most of these enzymes are multidomain proteins composed of different functional domains connected by linker peptides. Thus, it is very important to determine structural organization of glycoside hydrolases in terms of positions and orientations of their individual domains and comprehend the interplay between their multiple domains under close-to physiological conditions. To study the glycoside hydrolases, we propose to use small-angle X-ray and neutron scattering (SAXS and SANS) methods. Currently, the conformation, dynamics and function of GHs with multiple domains are not fully understood. This is why the information on their structural organization and mobility; mutual position and orientation of the individual domains and conformational changes induced by interaction with the substrates or changes in biochemical conditions (pH and T) might be very informative. Therefore, the main objective of the project is to conduct studies of glycoside hydrolases in solution, using SAXS and SANS. We would like to understand better interplay between conformation and flexibility of full-length GHs in solution and interactions between their individual domains in the context of their activity toward polymeric substrates. A large number of GHs have been cloned and expressed in the group of the Superviser, Prof. Dr. Igor Polikarpov (Molecular Biotechnology group, IFSC/USP) and we will follow already established protocols for their expression and purification. In SAXS experiments will be carried out in collaboration with Prof. Aldo Craievich (IF/USP) and Prof. Mario de Oliveira Neto (UNESP- Botucatu). Additionally, when judged necessary, the enzymes will be examined with SANS technique. The PhD candidate of this project has already an existing experience with SAXS and SANS methods and conducted experimental studies at several international scientific centers. We believe that this will be very beneficial for successful development of the present proposal. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

PIIADOV, VASSILI; DE ARAUJO, EVANDRO ARES; OLIVEIRA NETO, MARIO; CRAIEVICH, ALDO FELIX; POLIKARPOV, IGOR. SAXSMoW 2.0: Online calculator of the molecular weight of proteins in dilute solution from experimental SAXS data measured on a relative scale. Protein Science, v. 28, n. 2, p. 454-463, FEB 2019. Web of Science Citations: 7.

DE ARAUJO, EVANDRO ARES; MANZINE, LIVIA REGINA; PIIADOV, VASSILI; SEIKI KADOWAKI, MARCO ANTONIO; POLIKARPOV, IGOR. Biochemical characterization, low-resolution SAXS structure and an enzymatic cleavage pattern of BlCel48 from Bacillus licheniformis. International Journal of Biological Macromolecules, v. 111, p. 302-310, MAY 2018. Web of Science Citations: 1.