determination of ubiquitin dimerization kinetics via uv spectrophotometry n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Determination of ubiquitin dimerization kinetics via UV spectrophotometry PowerPoint Presentation
Download Presentation
Determination of ubiquitin dimerization kinetics via UV spectrophotometry

Loading in 2 Seconds...

play fullscreen
1 / 14

Determination of ubiquitin dimerization kinetics via UV spectrophotometry - PowerPoint PPT Presentation


  • 130 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Determination of ubiquitin dimerization kinetics via UV spectrophotometry' - lindsey


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.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
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
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
ubiquitin
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
cracking the ubiquitin code
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.

ubiquitin cellular signals
Ubiquitin Cellular Signals

F. Ikeda, I. Dikic. EMBO Reports. 2008. 9, 536–542

slide6
Goal
  • Is the distribution of linkages due to:
    • Specificity of E3 ligases

OR

    • Favorable interactions between monomers
      • Promiscuous ligases
linkage formation in vivo
Linkage formation in vivo
  • Reaction between:
    • C-terminal carboxyl
    • Amine of Lys side chain
      • Seven lysine residues
  • In vivo catalyzed by E3 ligases
in vitro representation of dimerization
In vitro representation of dimerization

UbwithC-terminal

cysteine

Ub76 K C variant

Native Dimer

synthesizing ub with a c terminal cysteine
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)

synthesizing ub k c variants
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)

results
Results
  • Initial reaction velocity (ν0)
  • K11C, K27C,
  • and K63C at
  • too low of a
  • concentration
results1
Results
  • ν0=k[A0][B0]
    • [A0] and [B0]
      • Considered constant (<5% decrease in reactants)
      • Measured initially with DTNB assay
  • Calculate rate constants (k)
future plans
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
acknowledgements
Acknowledgements
  • Prof. Ronald T. Raines
  • Dr. Langdon J. Martin
  • Kristen Andersen
  • The Raines Lab