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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

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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
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