Single-molecule studies of GroEL

Chaperonins are barrel-like oligomeric molecular machines which assist protein folding in the cell.
GroEL, the E. Coli Chaperonin, is made up of two homoheptameric rings, stacked back-to-back, with a cavity at each end in which protein folding can take place.
Protein folding by GroEL is mediated by a series of allosteric transitions driven by the binding and hydrolysis of ATP. GroEL binds ATP in a cooperative manner and there is evidence indicating that the conformational changes in GroEL take place in a concerted manner, i.e. all seven subunits of each ring perform synchronized motion.
The conformational cycling in GroEL is essential for its function. Nevertheless full elucidation of the reaction cycle of this fascinating molecular machine is elusive. In our group we are developing techniques that will allow direct observation of this protein at work.
 

Optical switch as a reporter for conformational changes

Using the crystal structure of GroEL captured at different stages of the reaction cycle we were able to engineer an optical switch into the structure. The switch is based on the quenching between a fluorescent label and tryptophan. The switch was designed by structural analysis so that quenching will occur in one conformation (fig. 1a) but not in the other (fig. 1b). This allowed following the structural changes of the protein by monitoring the fluorescence changes of a bright and stable visible-range fluorophore.

Figure 1: The optical switch in GroEL molecules. (a) The fluorescence of the dye (red) is quenched by the amino acid tryptophan (violet) in the apo conformation of GroEL. (b) In the ATP binding conformation, the quenching is removed. The structures in (a) and (b) correspond to two adjacent monomers in the trans and cis rings of the GroEL-GroES-ADP7 complex, respectively.

Switch-bearing molecules were found to have similar thermodynamic characteristics as the unmodified protein and demonstrated large ATP-dependent fluorescence intensity changes.

Single molecule studies of the conformational changes of GroEL

Using the above-mentioned switch, we are trying to monitor the conformational changes of the protein on the single molecule level. Such measurements are expected to give a direct observation on the folding cycle of GroEL.
In order to follow each molecule for a prolonged time, these experiments are conducted on surface tethered molecules. The molecules are excited by an evanescent wave produced by total internal reflection. The shallow profile of excitation resulting from the evanescent wave reduces the background thereby allowing the detection of the weak signal from single molecules. These signals are picked up by an ultra sensitive EM-CCD camera.
Figure 2 shows a typical microscope field before (a) and after (b) addition of 1 mM ATP. A strong increase in the fluorescence of many of the molecules is readily observed. The temporal trajectory of the fluorescence of one of these (circles in Figures 2a and 2b) is shown in Figure 2c.

Figure 2: Activity of the optical switch in labeled protein molecules deposited on a glass surface, monitored at the single molecule level. Changes in fluorescence after addition of (a) buffer only and (b) 1 mM ATP. (c) Temporal trajectory of the molecule marked with a red circle in (a) and (b).

Structural fluctuations of GroEL

Fast structural dynamics of proteins is studied by a home built Fluorescence Correlation Spectroscopy (FCS) system. FCS is a spectroscopic method suitable for kinetic measurements on a wide time-scale. While classical bulk kinetic methodologies follow the system as it approaches equilibrium after an abrupt change in conditions, FCS measurements are conducted under equilibrium conditions. In FCS measurements, kinetic data is extracted by monitoring the spontaneous fluctuations fundamental to all systems in dynamic equilibrium. Fluctuations in a thermodynamic system are inversely proportional to the number of particles in the system (N). The signal in FCS is a result of the autocorrelation of the time-fluctuations in fluorescence intensity which are inversely proportional to N. Therefore, in order to have detectible signals FCS measurements are conducted on a dilute sample (typically several nanomolars) in a small volume (diffraction limited spot in a confocal setup).
Using this system we are able to monitor fast structural fluctuation in GroEL. These measurements constitute the first direct experimental evidence for such fluctuations which were predicted by several theoretical groups. Singular value decomposition of the FCS curves of the protein under different conditions indicates that all the FSC curves in this system are a result of the weighted sum of two FCS curves indicating that the system fluctuates between two distinct states.