Biochemistry Poster Presentations

Towards the Crystallographic Structures of PhyA (Mitsoukella multacida) in Complex with Myo-Inositol Polyphosphates

Bruder, Lisza

  • Lisza Bruder, R. J. Gruninger, L. B. Selinger*, S. C. Mosiman*
  • Organization: University of Lethbridge
  • Funding: NSERC, AIF, CFI

Abstract: Myo-inositol polyphosphates (IPPs) are found in every domain of life, have a central role in eukaryotic cellular signalling and varied biological function including microbial pathogenesis. Phytases are enzymes that catalyze the stepwise removal of phosphates from the most abundant IPP, myo-inositol 1,2,3,4,5,6-hexakisphosphate (Ins P6). Recently, a fourth class of phytase (protein tyrosine phosphatase-like phytases or PTPLPs) was discovered in obligate anaerobes of the rumen and subsequently identified in diverse prokaryotes. PTPLPs have a comparatively highly-ordered sequential hydrolysis pathway and are likely the best candidates for engineering enzymes with novel IPP specificities. PhyAmm (PhyA from Mitsuokella multacida) contains a tandemly repeated PTPLP fold and has one of the highest known phytase activities. The structure of phyAmm has been determined at 2.3 A and the two active sites (D1 and D2) possess different IPP specificities. We will determine the X-ray crystallographic structures of inactive phyAmm mutants in complex with different naturally occurring substrates. These studies will provide the structural information required to engineer mutant enzymes capable of specifically dephosphorylating Ins P6 to any desired IPP.

Structural and Functional Properties of the Ribosome-Bound ATPase RbbA in Escherichia coli

Coatham, Mackenzie

  • MacKenzie Coatham, Jeffrey Fischer, Hans Joachim Wieden*
  • Organization: University of Lethbridge
  • Funding: UofL, NSERC, CFI

Abstract:

The process of protein synthesis is highly conserved across all domains of life. The synthesis of proteins is catalyzed by numerous factors that transiently bind to the ribosome during the various phases of translation. Many of these translation factors bind and hydrolyze GTP. These proteins are structurally related to the family of ATP hydrolyzing enzymes. The role of ATPases during translation is poorly understood and has not been studied extensively. ATP-dependent translation has been reported to be crucial for eukaryotic protein synthesis in the yeast elongation factor eEF-3 by releasing deacyl-tRNA from the ribosome upon the formation of peptide bonds and translocation (1). Here we report the purification and refolding from inclusion bodies of two yhih gene truncations, homologous to eEF-3, that encode for 65 kDa and 51 kDa Escherichia coli ATP binding proteins, deltaP541 and deltaL432 RbbA. For deltaP541 RbbA, we have estimated adenine nucleotide binding affinity and observed that ATPase activity was stimulated by 30S ribosomal subunits. For both of the yhih gene truncations, we have also generated mutants in the phosphate-binding loop of the ABC domain 2 that have been shown in eEF-3 to inhibit binding of ATP to the protein (2). This represents the first step in determining the functional role of ATP hydrolysis by the tandem ABC repeats in RbbA during translation.

(1)J. Xu, M. C. Kiel, A. Golshani, J. G. Chosay, H. Aoki, and M. C. Ganoza. Molecular localization of a ribosome-dependent ATPase on Escherichia coli ribosomes. Nucleic Acids Res. 2006; 34(4): 1158–1165.

(2)Yang H., Hamada K., Terashima H., Izuta M., Yamuguchi-Sihta E., Kondoh O., Satoh H., Miyazaki M., Arisawa M., Miyamoto C., Kitada K.. A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3. Biochim Biophys Acta. 1996; 1310(3): 303-8.

Small Molecular Chaperones for beta-glucocerebrosidase

Dhammi, Jennifer

  • Jennifer Dhammi, Ethan Goddard-Boger*, Steve Withers*
  • Organization: University of British Columbia
  • Funding: UBC

Abstract:Gaucher’s disease (GD) is a glycolipid lysosomal storage disorder caused by low activity of beta-glucocerebrosidase (GCase). GCase hydrolyzes glucosylceramide into ceramide and glucose in the lysosome. The most adverse effect of GD is early death. Current treatments are either expensive (enzyme replacement therapy) or have undesirable side effects (substrate reduction therapy). Those with central nervous system (CNS) complications are currently untreatable. Chaperone mediated therapy (CMT) may become a safe and economical way to relieve GD symptoms (including CNS impairments). CMT requires the mutant GCase to be able to exist in a properly folded conformation with catalytic activity. CMT involves GCase inhibitors acting as chaperones, which stabilize mutant GCases outside the lysosome by binding to them. Stable inhibitor-enzyme complexes are transported to lysosomes rather than being degraded. Substrate binding and the low lysosomal pH stabilizes mutant GCases. C-alkylated iminosugars appear to be the most potent GCase inhibitors. The synthesis of C-alklyated iminosugars is difficult and time consuming. A divergent synthetic scheme was proposed as a route to make a variety of GCase inhibitors easily and efficiently. Six inhibitors were synthesized and the KI values were determined for three of them, from which the most potent is (2R,3S,4S,5R) 2-[(2-Ethylsulfanyl)ethyl]-3,4,5-trihydroxy-piperidine hydrochloride ( KI=94-+5nm).

Nucleotide Dependence of HflX binding to the Escherichia coli Ribosome

Eagle-Bear, Shey

  • Shey Eagle-Bear, Hans Joachim Wieden*
  • Organization: University of Lethbridge
  • Funding: UofL - Chinook

Abstract:

HflX is a universally conserved GTPase, and is present in all three domains of life. GTPases are molecular switches located in the cell, and they are responsible for the regulation of many key processes in the cell (1). The cellular function of HflX is still unknown, and it is likely to interact with the ribosome; thereby helping to regulate translation. Previous studies have shown that HflX interacts with 50S ribosomal subunits (2) and that its GTPase activity is efficiently stimulated by the 70S ribosome (3). The purpose of this project was to further investigate the interaction between HflX and the ribosome. To this end we have reconstituted E. coli ribosome HflX complexes in different nucleotide bound states. Complexes were formed for 70S, 50S, and 30S ribosomal subunits, and subsequently purified using microultracentrifugation and a sucrose cushion to separate ribosomal subunits, free HflX, and the access of different nucleotides.

Trypsin digests were used to test whether different conformations of HflX exist on the ribosome when bound to different nucleotides. Trypsin is a serine protease that cleaves peptide bonds on the carboxyl terminus of arginine and lysine residues. Depending on the sites available for cleavage and their accessibility, the protein will be cut up into different sized fragments. Monitoring the time dependence of fragment formation will allow for the detection of different conformations of HflX in the respective ribosomal complexes.

1. Caldon, C.E. , and March, P.E. (2003). Function of the Universally Conserved Bacterial GTPases. Cur. Op. in Micro. 6, 135-139.

2. Jain, N., et al. (2009). E. coli HflX interacts with 50S ribosomal subunits in the presence of nucleotides. Biochem. Biophys. Res. Commun. 379, 201-205.

3. Shields, M.J., Fischer,J.J., and Wieden, H.J. (2009). Toward Understanding the Function of the Universally Conserved GTPase HflX from Escherichia coli: A Kinetic Approach. Biochem. 48, 10793-10802.

Kinetic Scheme of Pseudomonas aeruginosa EF-Tu; Guanine Nucleotide Exchange with the help of EF-Ts

Greeff, Kate

  • Kate Greef, Evelina deLaurentiis, Hans-Joachim Wieden*
  • Organization: University of Lethbridge
  • Funding: NSERC, AHFMR, AIF, CIHR, UofL

Abstract:

Pseudomonas aeruginosa (P.aeruginosa) is a bacterial pathogen which causes disease in humans, plants, and animals.

Elongation Factor Tu (EF-Tu) is a translational enzyme which is essential across all domains of life. During the translation process, EF-Tu functions to transport aminoacyl-tRNAs to the ribosome in a GTP bound conformation. Once correct codon recognition is made between aminoacyl-tRNA and mRNA, EF-Tu hydrolyzes GTP to GDP, leading to the release of aminoacyl-tRNA. Exchange Factor Ts (EF-Ts) is required in order for GDP to be exchanged for a new GTP molecule. By understanding the mechanism and kinetics of EF-Tu of P.aeruginosa and its role during translation, there may be new prospects for antibiotics which target this enzyme.

In order to study the guanine nucleotide binding kinetic scheme of EF-Tu from P.aeruginosa, rapid kinetics employing a Stopped Flow apparatus was used to solve this scheme. This utilized the property of Fluorescence Resonance Energy Transfer (FRET) in order to measure the guanine nucleotide association and dissociation rate constants to EF-Tu.


Recombinant Protein Expression in E. coli RelSeqNH385 Production of pppGpp

Pepneck, Tracy

  • Tracy Pepneck, Alvin Altimirano, Hans-Joachim Wieden*
  • Organization: University of Lethbridge
  • Funding: UofL

Abstract: The protein RelSeqNH385, the truncated version of RelA, was overexpressed and purified. This purified protein was then used in order to produce pppGpp, which is suspected to interact with GTPases, due to its involvement in the stringent response. The pppGpp that was produced was then purified and isolated from all other reaction components in order to study its binding capabilities. The pppGpp is produced by the cell using GTP and ATP during times of nutrient deprivation in order to limit the production of stable RNA's and to increase the amino acid production of the cell. The pppGpp that was produced in this study can be used to study if pppGpp does in fact interact with known GTPases. It can further be used to study suspected GTPases, and whether they interact with pppGpp. The interactions of pppGpp to these suspected GTPases are comparable to interactions with ADP and GDP.