Fig. 4
From: Protein degradation kinetics measured by microinjection and live-cell fluorescence microscopy
Site-specific coupling to small-molecule chromophores as reporters for protein degradation. (a) A model describing total cell fluorescence (TCF) changes as a result of protein degradation and subsequent cellular export of fragments. P refers to the labeled protein, F to the labeled degradation fragments, k1 to the degradation rate constant and k2 to the overall export rate constant. (b) TCF of single cells upon microinjection of dyes containing a maleimide group, quenched before injection with excess β-mercaptoethanol. The black line indicates the averaged degradation rate from an exponential decay model with a non-zero plateau parameter. (c) eGFP degradation was followed simultaneously by its intrinsic fluorescence and by an attached dye (indicated next to the orange arrow). eGFP fluorescence as a function of time is shown with dashed blue lines, while solid orange lines indicate dye signal. Black lines indicate simulated signal changes for a cell with averaged degradation rate (dashed, eGFP fluorescence; solid, respective dye fluorescence). The simple exponential decay model was used, except for eGFP coupled to Cy5-4 and co-injected with a bioPROTAC (see main text), where a free plateau parameter was introduced for fitting. Top row, Cy5-2; middle row, Cy5-4; bottom row, TMR. (d) Correlation between the degradation rate determined by loss of GFP fluorescence and from loss of fluorescence of small-molecule dyes attached to GFP. A perfect correlation would be indicated by the dashed grey line going through zero, while the linear regression line is shown in black. Top row, Cy5-2; middle row, Cy5-4; bottom row, TMR. (e) Simulated TCF data assuming a fixed degradation rate constant of a protein with a 30 min half-life and a range of dye export rates. Top, actual kinetics; bottom, apparent kinetics, if the observed traces are normalized at the first measured point after 0.5 h. (f) The difference between true and fitted simulated data using a first order exponential decay model is plotted against the dye export rates. The result of fitting with the exclusion of the first 30 min after injection is indicated in black, whereas fitting of data from the injection start is shown in grey. (g) Measured dye export rates. (h) Bar heights indicate averaged degradation rate constants based on eGFP (blue, left bar) or dye signal (orange, right bar); “Degr.” denotes bioPROTAC (see main text). (i) Calculated partition coefficient (cLogP) versus observed dye export rate.
