Quiros, Veronica (2010) Exploring the role of the H4-S4 loop and of the N-terminal His-tag in stabilizing quaternary structure of enolase from Streptococcus pyogenes. Masters thesis, Concordia University.
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Yeast (Saccharomyces cerevisiae) enolase is known to be a dimer of identical monomers while Streptococcus pneumoniae enolase is a tetramer of dimers. Dimeric enolase is a well-studied ubiquitous protein; however, not much is known about the octameric protein. Therefore, understanding the amino acids that determine quaternary structure could provide important information regarding its role. Based on the crystal structure of both enolases, it was hypothesized that the loop connecting helix 4 (H4) and strand 4 (S4) could be responsible for preventing formation of octamers in yeast enolase (Ehinger et al., 2004). In the octameric enolase this loop contributes to the dimer-dimer interface; however, in yeast enolase this loop is shorter due to the presence of an extra turn at the end of H4. The goal of my project is to understand if the extra turn at the end of H4 will destroy the dimer-dimer interface and prevent the formation of octameric enolase.
In order to study the effects of the extra turn in the octameric enolase, site-directed mutagenesis was performed on the H4-S4 loop. Residues 135-138 were changed from GGFN to ADLS, which corresponds to the amino acid sequence of yeast enolase. The same amino acids were also changed to AAAA, since alanine is known as a helix-forming amino acid. Characterization of the quaternary structure by analytical ultracentrifugation (AUC) showed that the variant enolase proteins are formed by a mixture of octamers, monomers, dimers and possibly other intermediates. Consistent with this observation, the specific activity of the variant proteins decreased considerably compared to the wild-type, due to a lower percentage of octamers. These results suggest that those amino acids in the extra turn play an important role in quaternary structure.
The second goal of this project is to determine if the polyhistidine tag (His-tag) affects the stability of octameric enolase. This protein is expressed with an N-terminal polyhistidine tag (His-tag) to facilitate the purification processes. The enolase gene also was inserted into a vector which carries a C-terminal His-tag with a thrombin recognition site, permitting removal of the tag. In order to study stabilization, sodium perchlorate, a weak chaotrope, was used to dissociate the octameric enolase (Karbassi et al., 2010). This dissociation was monitored by sedimentation velocity using AUC. Although, His-tagged proteins are commonly used to facilitate purification, we have observed that the His-tag at the N-terminus stabilizes the octameric structure.
|Divisions:||Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry|
|Item Type:||Thesis (Masters)|
|Degree Name:||M. Sc.|
|Thesis Supervisor(s):||Kornblatt, Judith|
|Deposited By:||VERONICA QUIROS|
|Deposited On:||13 Jun 2011 09:14|
|Last Modified:||13 Jun 2011 09:14|
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