Protein-binding dynamics imaged in a living cell

Abstract

Historically, rate constants were determined in vitro and it was unknown whether they were valid for in vivo biological processes. Here, we bridge this gap by measuring binding dynamics between a pair of proteins in living HeLa cells. Binding of a β-lactamase to its protein inhibitor was initiated by microinjection and monitored by Förster resonance energy transfer. Association rate constants for the wild-type and an electrostatically optimized mutant were only 25% and 50% lower than in vitro values, whereas no change in the rate constant was observed for a slower binding mutant. These changes are much smaller than might be anticipated considering the high macromolecular crowding within the cell. Single-cell analyses of association rate constants and fluorescence recovery after photobleaching reveals a naturally occurring variation in cell density, which is translated to an up to a twofold effect on binding rate constants. The data show that for this model protein interaction the intracellular environment had only a small effect on the association kinetics, justifying the extrapolation of in vitro data to processes in the cell.

Fig. 1.

FRET as a probe for TEM1-BLIP binding. (A) In vitro emission spectra of 1 μM CyTEM mixed with 1 μM YBLIP (red) shows reduced CyPet emission (peak at 476 nm) and elevated YPet emission (peak at 530 nm) compared to a mixture of CyPet and YPet (purple) at the same concentration. Competition with 10 μM TEM1 (black) or BLIP (cyan) abolishes FRET, whereas 10 μM BSA (green) had no effect on the interaction. (B) In-cell emission spectra of CyTEM mixed with YBLIP. A mixture of 2.5 μM from each protein was loaded to a capillary and injected into cells (red). Addition of 50 μM TEM1 to the mixture abolishes FRET (black). Spectra were normalized by the protein concentration in the cell. (C) In vitro binding curves of 0.1 μM CyTEM with 0.5 μM YBLIP probed either by FRET (black, 435-nm excitation, 515-nm cutoff emission) or by Tryptophan fluorescence (red, 280-nm excitation, 320-nm cutoff emission).

Fig. 2.

Association rate measurements in living cells. (A) Donor, FRET, and transmission channels are shown before and at 1 and 30 s after YBLIP injection. The reduction in the donor channel intensity 1 s after injection results from a twofold dilution of the cytoplasm. The complete time sequence of this cell is shown in Fig. S2 and in Movie S1. (B) Mean fluorescence intensities of the region indicated in red (A, Top Left) imaged by the donor, FRET, and acceptor channels. (C) Corrected FRET was calculated from the data shown in B using Eq. 2, and was subjected to numerical analysis (red). Protein concentrations of 0.9 μM CyTEM and 0.5 μM YBLIP were determined from the calibration curve shown in D, yielding an association rate constant of 1.6 × 10⁵ M^-1 s^-1. Corrected FRET of a control cell expressing 0.6 μM CyTEM and injected with 1 μM YPet is shown in blue. (D) Calibration curve of YBLIP (red) and CyTEM (green) fluorescence using purified proteins diluted into cell extract. (E) Normalized binding curves of WT and mutant complexes. CyTEM and YBLIP concentrations were 0.9 and 0.5 μM for the WT complex, 0.55 μM of both proteins for the fast mutant (YBLIP^D163K), and 1 μM of both proteins for the slow mutant (CyTEM^R243A; see also Movie S2). Solid lines are the fits produced by Pro-K II.

Scheme 1.

Fig. 3.

Binding rate measurements in HeLa cell extract. (A) Normalized association curves for the WT (0.5 μM of each protein) and the D163K mutant (0.2 μM of each protein) complexes. Solid lines are the fits produced by Pro-K II. (B) Normalized dissociation curves for the TEM1^E104A-YBLIP^WT complex in vitro and in cell extract. Solid lines are single exponent fits.

Fig. 4.

Association rate constants in living cells. (A) Association rate constants plotted versus the product of CyTEM and YBLIP concentrations in individual cells. (B) Pseudo-first-order kinetic analysis for CyTEM^R243A interacting with YBLIP^WT in cells. Observed rates of association are plotted against the YBLIP concentration in each cell, with the slope being the association rate constant.

Fig. 5.

The effect of cellular dilution on association rate constants. Dilution by microinjection was calculated as the ratio of CyTEM intensity before and after injection. (A) Dilution versus association rate constants in single cells. (B) Effect of low (1–1.4) and high (1.4–4) dilutions following PBS injections on t_1/2 of single cells as determined by FRAP. (C) Effect of low and high dilutions on association rate constants (data from A). The ratio of t_1/2 before and after injection was determined for each cell, and the average ratio for low- and high-diluted cells is shown in the inset. Error bars represent the standard error of the mean.