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| Author(s): |
Danielle Karoline Silva do Vale Castro
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: | 2018-07-30 |
| Examining board members: |
Richard Charles Garratt;
Alessandro Silva Nascimento
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| Advisor: | Richard Charles Garratt |
| Abstract | |
The septin family of proteins is characterized by their ability to bind guanine nucleotides and associate into filaments. Several biological functions have been reported for these filaments and their dissociation may be related to pathologies. Human septin is 12 specifically expressed in testes and has been identified in filaments that form the sperm annulus, whose integrity is related to its morphology. Although many studies have been reported, the molecular and physiological bases of septin filament function and self-assembly have yet to be completely elucidated. This study aims to obtain structural information for the nucleotide binding domain of SEPT12 (SEPT12G), the SEPT12GT89M mutant and the SEPT7-SEPT12 heterodimer. Expression of these proteins was performed in E. coli Rosetta(DE3) strain using the pET28a (+) and pETDuet-1 expression vectors. Purification was performed by affinity and size exclusion chromatography. The SEPT12G protein was submitted to an evaluation of its oligomeric state, intrinsic fluorescence, nucleotide content, GTPase activity and thermal transition. The oligomeric state and nucleotide content of SEPT7-SEPT12 was also evaluated. Crystallization assays were performed for all proteins. Data collection on line I24 of the Diamond Light Source (Didcot, England) resulted in high-resolution data sets for SET12G and SEPT12GT89M but only low resolution data for the SEPT7NGc. Biophysical studies showed that SEPT12G was obtained in its native form or, in other words, capable of binding and hydrolyzing GTP and that the purified heterodimer presented both proteins. The crystallographic structures were solved by molecular replacement allowing the identification of features characteristics of the group I septins (SEPT3, SEPT9 and SEPT12). The structure also confirmed that all the proteins of this group are able to form two different NC interfaces: open and closed. In addition, it reinforced the observation that the α5\' helix assumes a different orientation, whose function has not yet been clarified, but without doubt is a characteristic of this group which may be related to anchoring the polybasic regions whilst in the open conformation. The SEPT12T89M mutant crystal structure shows that the first shell coordination of the Mg2+ ion is altered, leading to an interruption of the universal switch mechanism and a consequent lack of catalytic activity. Finally, structural studies of the interaction between SEPT12 and SEPT7 were not possible, since all attempts resulted in crystals containing only SEPT7. This may be a consequence of SEPT12 precipitation or the crystallization condition used, destabilizing the heterodimeric interface. (AU) | |
| FAPESP's process: | 16/19734-2 - Molecular recognition in septins: the interface studies between SEPT7 and SEPT12 |
| Grantee: | Danielle Karoline Silva Do Vale Castro |
| Support Opportunities: | Scholarships in Brazil - Master |