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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

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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


Determination of ubiquitin dimerization kinetics via uv spectrophotometry

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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