1 / 14

Determination of ubiquitin dimerization kinetics via UV spectrophotometry

Determination of ubiquitin dimerization kinetics via UV spectrophotometry. Joel Prince Advisor: Ronald T. Raines Dept. of Biochemistry, University of Wisconsin–Madison. Outline. Background Ubiquitin Ubiquitin code and signals Research goal Experimental Approach

lindsey
Download Presentation

Determination of ubiquitin dimerization kinetics via UV spectrophotometry

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Determination of ubiquitin dimerization kinetics via UV spectrophotometry Joel Prince Advisor: Ronald T. Raines Dept. of Biochemistry, University of Wisconsin–Madison

  2. Outline • Background • Ubiquitin • Ubiquitin code and signals • Research goal • Experimental Approach • Synthesizing Ub with C-terminal cysteine • Synthesizing Ub K C variants • Results • Kinetics plot • Future Plans

  3. Ubiquitin Yeast/Human Ubiquitin (1UBQ) • Highly conserved (ubiquitous) in eukaryotes • Important post-translational modification • > 4% of genes in the human genome encode proteins involved in ubiquitin signaling

  4. Cracking the Ubiquitin Code Heterotypic Polyubiquitination Monoubiquitination S S S HomotypicPolyubiquitination with respective % linkages in vivo S S S S K11 (28.0%) K27 (9.0%) K6 (10.9%) K29 (3.2%) S S S S K48 (29.1%) K33 (3.5%) K63 (16.3%) Xu, P. et. al. J. Cell.2009. 137, 133–145.

  5. Ubiquitin Cellular Signals F. Ikeda, I. Dikic. EMBO Reports. 2008. 9, 536–542

  6. Goal • Is the distribution of linkages due to: • Specificity of E3 ligases OR • Favorable interactions between monomers • Promiscuous ligases

  7. Linkage formation in vivo • Reaction between: • C-terminal carboxyl • Amine of Lys side chain • Seven lysine residues • In vivo catalyzed by E3 ligases

  8. In vitro representation of dimerization UbwithC-terminal cysteine Ub76 K C variant Native Dimer

  9. Synthesizing Ub with a C-terminal cysteine • Quikchange mutagenesis • Add 77th residue: cysteine • NTB-protected thiol • Important for colorimetric analysis DTNB NTB-protection Displacement via thiol NTB (412 nm)

  10. Synthesizing Ub K C variants • Quikchange mutagenesis • Site-directed technique • Mutate Lys 6, 11, 27, 29, 33, 48, and 63 • Result in cysteine mutants • Reactive thiol necessary for displacing NTB NTB (412 nm)

  11. Results • Initial reaction velocity (ν0) • K11C, K27C, • and K63C at • too low of a • concentration

  12. Results • ν0=k[A0][B0] • [A0] and [B0] • Considered constant (<5% decrease in reactants) • Measured initially with DTNB assay • Calculate rate constants (k)

  13. Future Plans • Analyze kinetics of reaction of DTNB and Ub K C variants • Hypothesis: observation of similar rate constants • Re-express oxidized variants • Remedy observed linear kinetic trends • Compare kinetics to thermodynamic

  14. Acknowledgements • Prof. Ronald T. Raines • Dr. Langdon J. Martin • Kristen Andersen • The Raines Lab

More Related