Biochemistry Oral Presentations

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.


Purification and Characterization of Tocopherol Cyclase

Hrabowych, Alexandra

  • Alexandra Hrabowych, George Oriss, Joerg Stetefeld*
  • Organization: University of Manitoba
  • Funding: UofM, NSERC

Abstract: Vitamin E is a fat soluble antioxidant which is synthesized exclusively by photosynthetic organisms and is an essential nutrient for both humans and animals, protecting the body from damaging free-radicals. A deficiency results in an increased risk of cancer and is correlated with other chronic diseases, such as heart failure. Vitamin E is the collective name for eight naturally occurring compounds, of which alpha-tocopherol has the highest biological relevance. The specificity for alpha-tocopherol is determined by the alpha-tocopherol transfer protein which transports Vitamin E to distant sites in the body. Tocopherol Cyclase is a 41.7 kDa, membrane-bound enzyme that is responsible for the enantioselctive cyclisation of 2,3-dimethyl-5-phytylhydroquinone to generate gamma-tocopherol, which is then methylated to give alpha-tocopherol. In organic synthesis the cyclisation results in a racemic mixture rendering half of the product biologically useless. Therefore, it is of great interest to understand how tocopherol cyclase is able to catalyze formation of the product with a 100% yield. Over the past year we have established a reproducible purification protocol for Tocopherol Cyclase from the cyanobacterium Anabaena sp. We hope to characterize this protein through structure-function studies, using X-ray crystallographic techniques. Currently we are able to grow and harvest stable crystals, but resolution is limited by the small size. Larger crystals have been isolated, but are anisotropic. Without homogenous diffraction in all directions, limited information can be obtained from this data. At present, we are working to optimize the crystallization conditions in hopes of improving crystal size and morphology.

In vitro investigation on the interaction between U24 from Herpes Virus Type 6 and T-cell proteins

Lin, Frances Yi-Shiuan

  • Frances Yi-Shiuan Lin, Andrew Tait, Susana Strauss*
  • Organization: University of British Columbia
  • Funding: UBC

Abstract: Myelin plays an essential role in the insulation of nerve axon where it acts to increase the rate of conduction between nerve cells. Autoimmune degradation of myelin is a typical symptom found in Multiple Sclerosis (MS). Possible causes of MS arise from genetic or environmental (possibly viral) factors. Research has shown Human Herpes virus type -6 (HHV-6) to be associated with at least a subgroup of MS patient. Although the exact mechanisms are unknown, viral infection may play a role in the pathogenesis of MS potentially through the mechanism of molecular mimicry. Interestingly, glycoprotein, U24 from HHV-6 shares a stretch of 7 similar amino acids sequence with Myelin Basic Protein (MBP 92-104=IVTPRTPPPSQGK; U24 1-13= MDPPRTPPPSYSE). The shared sequence suggests an immunological explanation combined with the concept of molecular mimicry to the clinical phenomenon, demyelination found in multiple sclerosis patients. Previously we have identified both U24 and MBP are targets for the protein kinase, ErK2 (MAPK). Here, we will identify the potential interaction between U24 from HHV-6a through in vitro pull down experiment followed by SDS-PAGE analysis. Finally, we demonstrated that U24 from HHV6 can interact with T-cell proteins in salt dependent manner and interactions are observed at low salt.

Monitoring Protein Folding Dynamics Using Single-Molecule Spectroscopy

Ma, Martha

  • Martha Ma, Hongbin Li*
  • Organization: University of British Columbia
  • Funding: NSERC, CRC, CFI, CHIR

Abstract: A class of proteins with high resistance to external forces, known as elastomeric proteins, possess high extensibility and elasticity, making them the object of particular interest as materials for the bottom-up construction of materials, such as those used in biomedical imaging, biosensing and drug delivery. To this end it is vitally important to study protein stability, which can be increased by binding with metal ions.1 Atomic force microscopy (AFM) plays an important role in elucidating the response of proteins to metal-ion binding, and can be used to further our ability to engineer proteins with tailored mechanical properties. My research was towards investigating the response to metal binding of two main types of proteins: engineered domain-insertion proteins, and a hetero-polyprotein with two distinct domains arranged in a tandem repeat. Fluorescence spectroscopy and stopped-flow measurements indicated an increase in stability of the domain-insertion proteins after Ni2+ binding. AFM measurements of the polyprotein yielded characteristic saw-tooth patterns (Figure 1), indicating unfolding events of metal ion-bound protein domains.


Utilizing a Novel Method of RNA Preparation to Isolate PKR Inhibitors

Munch, Garett

  • Garett Munch, Sean McKenna*
  • Organization: University of Manitoba
  • Funding: Canadian Health Research Council

Abstract: The costs related to producing many different RNA Samples, in both time and money, act as a deterrent for many projects involving a large number of RNA species. In order to reduce costs related to studying RNA, a novel method of RNA preparation has been developed. This method, done completely in vitro, reduces the costs involved with producing RNA, as well as allows samples to be produced in less than two days. A current project in the McKenna Lab involves assaying a number of double-stranded RNA inhibitors of PKR for both binding affinity and inhibitory activity. The inhibitor chosen for study is known as VA1 and is found in Adenovirus. A wild-type species will be used as a control, and other species generated will relate these changes in sequence to affinity and PKR inhibition. Here we show that the cell-free method of RNA production is capable of generating RNA samples, which are able to bind to PKR.

Expression and Purification of Protein Phosphatase I

Tyagunova, Yevgeniya

  • Yevgeniya (Jane) Tyagunova, Sean McKenna*
  • Organization: University of Manitoba
  • Funding: Manitoba Health Research Council

Abstract: A functional immune system that is able to recognize and eliminate a pathogen is quite important to a host cell's ability to resist infections. Pathogen sensors (proteins) can be expressed in multiple locations in a human system and there activation can lead to an innate immune system response. This generates specific signals or molecules that initiate signal transduction cascades to protect the host from the pathogen. The activation of PKR (protein kinase RNA-activated) is a very important cellular event that influences antiviral and cell growth pathways, and is involved in numerous antiviral mechanisms by shutting down the protein expression in the host. The mechanism of activation of PKR by viral RNA and the regulation of its activity in a healthy organisms system is not quite understood, and has been a popular research topic. In this presentation a possible protein inhibitor of PKR is proposed to be Protein Phosphatase 1. The expression in Escherichia coli and attempts at purification of this protein have been studied and will be presented in detail.

An Introduction to iGEM

Vigar, Justin

  • Justin Vigar, Lisza Bruder, Alix Blackshaw, Hans Joachim Wieden*
  • Organization: University of Lethbridge

Abstract: The emerging field of synthetic biology is the combination of engineering approaches with state of the art molecular and cellular biology techniques. The International Genetically Engineered Machines competition (iGEM) is an interdisciplinary undergraduate competition of teams with members from diverse backgrounds such as biology, chemistry, biochemistry, neuroscience, business, ethics, and new media. The undergraduate teams are given a kit of standard biological parts and spend a summer creating a newly developed genetic system while exploring the ethical implications. Created in 2004 with only 5 teams competing, iGEM has now grown to include over 120 teams with projects ranging from medical to environmental to computational applications. Competing against schools such as Harvard, MIT, Stanford, Queens, McGill and Berkely, the University of Lethbridge has proven that a small team of dedicated students can be successful internationally. They have received one bronze and two gold medals for their projects since joining iGEM in 2007. iGEM gives the opportunity for undergraduate students to improve their lab skills, develop cutting edge research and share scientific knowledge with other participants from around the world. A short summary of the history of iGEM and an outline of the University of Lethbridge’s current team’s project will be given.