ATP Dependent Rotational Motion of Group II Chaperonin Observed by X-ray Single Molecule Tracking

<div><p>Group II chaperonins play important roles in protein homeostasis in the eukaryotic cytosol and in Archaea. These proteins assist in the folding of nascent polypeptides and also refold unfolded proteins in an ATP-dependent manner. Chaperonin-mediated protein folding is dependent on the closure and opening of a built-in lid, which is controlled by the ATP hydrolysis cycle. Recent structural studies suggest that the ring structure of the chaperonin twists to seal off the central cavity. In this study, we demonstrate ATP-dependent dynamics of a group II chaperonin at the single-molecule level with highly accurate rotational axes views by diffracted X-ray tracking (DXT). A UV light-triggered DXT study with caged-ATP and stopped-flow fluorometry revealed that the lid partially closed within 1 s of ATP binding, the closed ring subsequently twisted counterclockwise within 2–6 s, as viewed from the top to bottom of the chaperonin, and the twisted ring reverted to the original open-state with a clockwise motion. Our analyses clearly demonstrate that the biphasic lid-closure process occurs with unsynchronized closure and a synchronized counterclockwise twisting motion.</p></div

Angular diffusion coefficient of the group II chaperonin in the tilting (θ) direction.

<p>The values were obtained from the slope of the MSD versus time plot (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064176#pone-0064176-g003" target="_blank">Figure 3</a>-C). The lines in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064176#pone-0064176-g003" target="_blank">Figure 3</a>-C were fitted with least-squares fitting to the following equation: <i>MSD = 4Dt</i>, where <i>MSD</i> is the mean square angular displacement, <i>D</i> is the angular diffusion constant, and <i>t</i> is time interval.</p

Schematic model of the conformational changes in the group II chaperonin.

<p>Schematic model of the conformational changes in the group II chaperonin.</p

ATP-dependent rotational motion of a group II chaperonin tracked by DXT.

<p>(A) Conformational changes of the group II chaperonin in the absence (left) and presence (right) of ATP. (B) Schematic illustration of the detection of internal motions of group II chaperonins by DXT. (C) Typical DXT traces of gold nanocrystals immobilized on the ring of the group II chaperonin in the absence (upper panel) and presence of ATP (lower panel). (D) The distribution of the absolute angular displacement of the group II chaperonin in the twisting (χ) direction. About 500 DXT trajectories are used to make histogram. The trajectories with an angular displacement greater than 30 mrad in the χ direction were counted as inset bar-graph.</p

ATP-triggered rotational analysis of group II chaperonins in the θ direction.

<p>(A) Time-series histograms of the absolute angular displacement in the θ direction per frame (36 ms). (B) Tryptophan fluorescence changes for a group II chaperonin (TKS1-Cpn L265W) in a mixture of ATP, as measured with a stopped-flow spectrofluorometer. (C) Mean square angular displacement (MSD) in the θ direction as a function of time interval in the presence of 0 mM ATP, 2 mM ATP, or 1 mM ATP-AlFx.</p