Figure 1. Final product distribution for SCF…
Figure 1. Final product distribution for SCF Cdc4 and CycE
a , In reaction 1, pre-incubated…
Figure 1. Final product distribution for SCFCdc4 and CycEa, In reaction 1, pre-incubated 32P-labeled CycE and SCFCdc4 were added to the charged E2 mix. In reactions 2 and 3, excess unlabeled CycE was pre-incubated with charged E2 mix and labeled CycE, respectively. b, The single encounter polyubiquitin chain length distribution, λCycE. Error bars: +/− SD, n=3. c, If η(1)=100%, then φ=λ. d, If φ(1)=100%, then η=λ. e, Deconvolution of λCycE and exponentially distributed η. f, Deconvolution of λCycE and normal distributed φ. g, Mass spectrometry of Cdc34 thioesterified for 2’ with indicated components. h, Simulated kinetics η(1)=100%. i, Simulated kinetics φ(1)=100%.
Figure 2. Millisecond kinetics of a single…
Figure 2. Millisecond kinetics of a single encounter reaction reveal sequential processivity
a , To…
Figure 2. Millisecond kinetics of a single encounter reaction reveal sequential processivitya, To achieve millisecond temporal resolution CycE reactions were performed on a quench flow apparatus and products were evaluated by SDS-PAGE and phosphorimaging. The reaction scheme matched reaction 2 of Fig. 1a. The asterisk marks a contaminant. Sn refers to CycE modified with n ubiquitins. b, Quantification shows successively longer lag phases for each additional ubiquitin added in the chain. The data was fit using closed form solutions refined by global regression analysis to a model with η=1. The error bars represent the range of values, n=2.
Figure 3. Human Cdc34-SCF β-TrCP is sequentially…
Figure 3. Human Cdc34-SCF β-TrCP is sequentially processive
a , same as Fig. 1a, except that…
Figure 3. Human Cdc34-SCFβ-TrCP is sequentially processivea, same as Fig. 1a, except that human Cdc34 and Nedd8-conjugated SCFβ-TrCP were assayed with 32P-labeled β-Cat substrate. b, Product distribution (λβ-Cat) was quantified as in Fig. 1b. Error bars: +/− SD, n=3. c, The poisson distribution of φ using λβ-Cat that deviated the most from φ(1)=100% within our set error bounds with α=0.2. d, β-Cat reactions with the scheme of reaction 2 (Fig 1a) performed on a quench flow apparatus. e, Quantification shows successively lengthening lag phases for each additional ubiquitin added in the chain. The data was fit as in Fig. 2b.
Figure 4. Kinetic basis for Cdc34-SCF processivity
Figure 4. Kinetic basis for Cdc34-SCF processivity
The millisecond kinetics of a single encounter reaction…
Figure 4. Kinetic basis for Cdc34-SCF processivityThe millisecond kinetics of a single encounter reaction were fit to a sequential model revealing estimates for individual transfer and dissociation rates for each intermediate in the generation of polyubiquitylated CycE (blue numbers) and β-Cat (red numbers) products. The percentages listed above or below each product were the percentages from the final product distributions (λ) shown in Fig. 1b and Fig. 3b.
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