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| Author(s): |
Caroline Hoff
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) |
| Defense date: | 2008-08-29 |
| Examining board members: |
Richard Charles Garratt;
João Alexandre Ribeiro Gonçalves Barbosa;
Frederico José Gueiros Filho
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| Advisor: | Richard Charles Garratt |
| Abstract | |
Septins are GTP-binding proteins. They were originally identified in fungi and act during the final stages of cell division. Subsequently, they were also identified in other eukaryotic with the exception of plants. Septins are purified from Saccharomyces cerevisiae, Drosophila, and mammalian brain in the form of heterofilaments and consist of three principal regions: a variable N-terminal domain, a central highly conserved GTP-binding domain and a coiled-coil domain at the C-terminus. It is known that there are at least 14 human septin genes but as yet, there is still relatively little structural information concerning their protein products. Of these, only three (septins 2, 6 and 7) have had part of their three-dimensional structure (principally the GTPase domain) determined by X-ray crystallography. The septin family can be divided into four subgroups on the basis of sequence similarity. One of them (group II) is composed of SEPT6, SEPT8, SEPT10, SEPT11 and SEPT14. SEPT11 was described for the first time in 2004 and was observed to be expressed in several human tissues. It is described as forming part of heterofilamentous complexes with other septins and may be involved in the glomerular filtration in the kidney. In order to characterize the SEPT11 protein, it was initially divided into its component structural domains and several constructs elaborated: SEPT11NG (N-terminal and GTPase domain), SEPT11G (GTPase domain), SEPT11GC (GTPase domain and C-terminal) and SEPT11NGC (N-terminal, GTPase and C-terminal domains). The genes corresponding to SEPT11G and SEPT11GC were cloned in an expression vector and SEPT11NG into a bacterial propagation vector. Both SEPT11G and SEPT11GC were successfully produced in E. coli and subsequently purified. Both circular dichroism spectra and computational techniques indicated that SEPT11 exhibited that both proteins were of the α/β type, as anticipated, coherent with the structure of SEPT6. Light scattering measurements at 350 nm showed that the protein undergoes a process of aggregation at temperatures above 30°C, similar to other septins (SEPT2 and SEPT4) and consistent with thermal stability studies using circular dichroism. Results of size exclusion chromatography indicated that SEPT11G formed dimers (similar to SEPT2, SEPT4 and SEPT7) and SEPT11GC apparently formed monomers only. All of these experimental data suggest that the heterologously expressed proteins described here folded into their native conformation. On the other hand, we also demonstrated that SEPT11G was nucleotide free even when purified in the presence of excess GTP or GDP. Homology modeling of the GTPase domain of SEPT11 failed to reveal any significant differences with respect to SEPT6 which would explain this lack of binding activity. We speculate that in the case of SEPT11 (and possibly other members of the group II septins) the presence of partner septins and the formation of the heterofilaments are essential for stable nucleotide binding. (AU) | |