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Review
. 2001 Jul;24(3):278-88.
doi: 10.1006/meth.2001.1188.

Application of fluorescence resonance energy transfer to the GroEL-GroES chaperonin reaction

H S Rye  1
Affiliations
Review

Application of fluorescence resonance energy transfer to the GroEL-GroES chaperonin reaction

H S Rye. Methods. 2001 Jul.

Abstract

Fluorescence resonance energy transfer (FRET) is a sensitive and flexible method for studying protein-protein interactions. Here it is applied to the GroEL-GroES chaperonin system to examine the ATP-driven dynamics that underlie protein folding by this chaperone. Relying on the known structures of GroEL and GroES, sites for attachment of fluorescent probes are designed into the sequence of both proteins. Because these sites are brought close in space when GroEL and GroES form a complex, excitation energy can pass from a donor to an acceptor chromophore by FRET. While in ideal circumstances FRET can be used to measure distances, significant population heterogeneity in the donor-to-acceptor distances in the GroEL-GroES complex makes distance determination difficult. This is due to incomplete labeling of these large, oligomeric proteins and to their rotational symmetry. It is shown, however, that FRET can still be used to follow protein-protein interaction dynamics even in a case such as this, where distance measurements are either not practical or not meaningful. In this way, the FRET signal is used as a simple proximity sensor to score the interaction between GroEL and GroES. Similarly, FRET can also be used to follow interactions between GroEL and a fluorescently labeled substrate polypeptide. Thus, while knowledge of molecular structure aids enormously in the design of FRET experiments, structural information is not necessarily required if the aim is to measure the thermodynamics or kinetics of a protein interaction event by following changes in the binding proximity of two components.

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Figures

FIG. 1
FIG. 1
Positions of the fluorophore labeling sites in the GroEL–GroES complex. The positions of the donor chromophore attached to GroEL (EDANS) and the acceptor chromophore attached to GroES (F5M) were modeled into the structure of the GroEL–GroES complex (16) using InsightII (Molecular Simulations, San Diego, CA). While the exact orientations of the dyes are only speculative, their effective separation is constrained in the GroEL–GroES complex to within 35–40 Å. The GroES subunits are colored light green and the GroEL subunits are colored purple. Inset: Wider view of the entire GroEL–GroES complex.
FIG. 2
FIG. 2
The protein-to-dye labeling ratio can be examined by denaturing ion exchange chromatography. A sample of EL315C labeled with IAEDANS was purified and then denatured in 8 M urea. This sample was injected onto a Mono Q 5/5 column equilibrated in buffer containing 6 M urea, and the column was developed with a linear NaCl gradient (see Materials and Methods). The elution positions of the unconjugated El315C subunits and those that have been labeled with IAEDANS (EL315-D) are indicated. Top: Observed absorbance at 229 nm; bottom: observed fluorescence at greater than 425 nm with excitation at 336 nm. Note that the protein peak corresponding to the EL315-D subunits is fluorescent. The labeling ratio indicated by the peak areas in the upper panel is 0.3, or approximately 4 dyes per tetradecamer on average.
FIG. 3
FIG. 3
The oligomeric structures of labeled GroEL and GroES introduce significant complexity in the donor–acceptor distance distribution. (A) Labeling of a 7-subunit ring produces a wide variety of conjugated species if the reaction does not proceed to completion. (B) The theoretical distribution of labeled species from a 14-subunit protein is plotted as a function of the number of labeled sites. The distribution of labeled species is assumed to be Poisson and plots are shown for average labeling of 1, 4, 8, 10, and 12 labels per oligomer. A sample of GroEL–GroES complexes created from incompletely labeled proteins would possess a similar distribution of donor-to-acceptor distances. (C) The rotational symmetry of the GroEL–GroES complex adds an additional layer of complexity to the donor–acceptor distance distribution.
FIG. 4
FIG. 4
The specificity of labeling is established by reversed-phase chromatography of a protease-fragmented sample. A sample of EL315C labeled with IAEDANS was digested with trypsin and then loaded onto a C18 reverse-phase column (see Materials and Methods). Top: Absorbance at 229 nm; bottom: fluorescence at greater than 425 nm with excitation at 336 nm. Note that a single peptide of the many generated by the EL315-D tryptic digest is fluorescent, indicating that the site of fluorophore conjugation is unique.
FIG. 5
FIG. 5
Changes in the fluorescence intensity of EL315-D andES98-A in the presence of ATP demonstrate that complex formation can be observed by FRET. Top: Fluorescence spectra of EL315-D (500 nM) and ES98-A (500 nM), both individually and mixed together. Bottom: Spectra of EL315-D and ES98-A mixed together in the presence and absence of 5 mM ATP. Note the decrease in fluorescence around 425 nm and the enhancement of fluorescence around 520 nm when ATP is added to the mixture, indicating the transfer of excitation energy from the IAEDANS donor to the fluorescein acceptor in the GroEL–GroES complex. For all spectra, the excitation wavelength was 336 nm, the temperature was maintained at 25°C, and the buffer used was 50 mM Hepes, pH 7.6, 5 mM KOAc, 10 mM Mg(OAc)2, 4 mM DTT.
FIG. 6
FIG. 6
Dissociation of ES98-A from EL315-D during steady-state ATP turnover. (A) Schematic of experiment in which EL315-D is first mixed with ES98-A and ATP and allowed to come to a steady state, after which the sample is rapidly mixed in the stopped flow with an excess of unlabeled GroES (gray disk). The loss of energy transfer as ES98-A is replaced by unlabeled GroES is monitored as an increase in the donor fluorescence intensity and a decrease in the acceptor fluorescence intensity. For this experiment, 188 nM EL315-D was premixed with 175 nM ES98-A and 5 mM ATP and loaded into one stopped-flow syringe. The steady-state mixture was then mixed (4:1) with 7.5 µM GroES in the stopped-flow. The rate of ES98-A release can be monitored as either the dequenching of the donor (B, C) or loss of fluorescence of the acceptor (D, E). Control experiments in which one of the energy transfer partners was replaced with its unlabeled conjugate are shown in (B) and (D), where “D” designates EL315-D alone, “A” designates ES98-A alone, and “D+A” indicates the use of donor and acceptor labeled proteins together. For the “D”-only trace, the initial steady-state mix was made with EL315-D and unlabeled GroES, with unlabeled GroEL used as the competitor. For the “A”-only trace, the initial steady-state mix was made with ES98-A and unlabeled GroEL, with unlabeled GroES used as the competitor. Note that the plots in (B) and (D) have been scaled and offset so that trends in the donor-only and acceptor-only traces can be visualized on the same graph with the donor plus acceptor trace. As a result, the changes in the “D” and “A” traces appear exaggerated relative to the “D+A” traces. The corrected FRET signals are shown in (C) and (E). In both channels, the data are fit by a single exponential model (shown as a thin white line superimposed on each relaxation curve: for (C), kslow = 0.031 ± 0.0001 s−1 and for (E) kslow = 0.029 ± 0.0001 s−1). The measured rate constant does not change at different ratios of ES98-A to EL315-D, is not dependent on the salt concentration (up to 250 mM KCl), and is independent of the concentration of competitor GroES used (not shown). The data for this figure were taken from (23) with permission. © Cell Press 1999.
FIG. 7
FIG. 7
Binding of ES98-A and Rubisco-A to EL315-D. (A) Schematic of binding experiments used to monitor the rate of ES98-A and dRub-A association with unliganded GroEL. The promotion of FRET as an EL315-D ring binds either ES98-A or dRub-A results in a decrease in the donor fluorescence. (B) Binding of ES98-A to EL315-D at equimolar concentrations. In the upper trace, ES98-A and EL315-D were mixed at final concentrations in the stopped-flow of 50 nM each, and in the lower trace at 1 µM; ATP in each case was 2 mM. The bimolecular rate constants derived from these traces are 5.7 × 107 and 2.9 × 107 M−1 s−1 for the 50 nM and 1 µM traces, respectively. (C) Binding of dRub-A to EL315-D. The upper curve was generated at final concentrations of 100 nM dRub-A and 100 nM EL315-D. The lower was produced at a concentration of 1 µM in each component. Bimolecular rate constants were estimated from the 100 nM traces only (k ~ 1 – 2 × 107 M−1 s−1), due to a competing aggregation reaction of the dRub-A at higher concentrations. The data for this figure were taken from (23) with permission. © Cell Press 1999.
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