Picometer-scale dynamical observations of individual membrane proteins: The case of bacteriorhodopsin

In vivo measurements of dynamical conformational changes in single biomolecules under functional conditions have had a tremendous impact on molecular and cell biology. However, even single-molecule fluorescent resonance energy transfer cannot easily monitor the intramolecular dynamics in cell systems due to shortcomings in monitoring precision. Here, we report dynamical observations of irreversible intramolecular conformational changes in a single-membrane protein [bacteriorhodopsin (BR)] using diffracted x-ray tracking. The light-driven proton pump BR is the best-characterized membrane protein. The position of BR’s 35th amino acid, which is located farthest from retinal, exhibits a momentary positional jump of $0.73\pm 0.48\mathrm{\AA }$ due to the expression of its function. Following that, we observed Brownian motion without the diffracted spots returning to their initial positions. The average width of this jump is about 14 times larger than that of thermal Brownian motion and agrees with estimated movements from known x-ray crystallography data. This result is an important step toward realizing in vivo observations of single-molecular conformational changes in membrane proteins.

Figure 1

Experimental designs of DXT. (a) Schematic drawing of the cross-sectional view of DXT in the case of BR S35C. DXT monitors the spots of diffracted x rays from individual nanocrystals that are tightly labeled with individual BR S35C. Samples containing the immobilized BR S35C were filled with the oxygen scavenger system ($25\mathrm{mM}$ glucose, $14\mathrm{mM}$ 2-mercaptoethanol, $10\mathrm{nM}$ catalase, and $2.5\mu \mathrm{M}$ glucose oxidase) in $50\mathrm{mM}$ MOPS $\left(\mathrm{pH}7.0\right)$. (b) Schematic illustration showing the top view of DXT using BR S35C. The 1:1 ratio of BR molecules to nanocrystals is possible because of the Hg compound (PCMB) first labeled with excess sulfhydryl groups in BR S35C. BR S35C-labeled PCMB is shown in silver, and nonlabeled BR S35C is shown in black; that is, black BR S35C can be linked to gold nanocrystals. (c) Optical-absorption spectrum of wild-type BR $\left(A\right)$, BR S35C $\left(B\right)$, colloidal gold $\left(C\right)$, colloidal gold-labeled BR S35C $\left(D\right)$, and calculated spectra $(B+C)$ $\left(E\right)$. Absorption maximum of $\left(A\right)$ or $\left(B\right)$ is $568\mathrm{nm}$.

Figure 2

Real-time tracking of diffracted spots and the view of positional jump of BR S35C due to light irradiations $(\sim 570\mathrm{nm})$. (a) DXT images of diffracted spots from the gold nanocrystal linked to individual BR S35Cs. The arrows show the positions of a diffracted spot at $36\text{−}\mathrm{ms}$ intervals. The significantly positional jump of a diffracted spot was observed by flashing light at a particular wavelength. (b) These trackings, which are randomly chosen from 70 observed spots, were measured from DXT images. The arrow shows the light irradiation at time 0 for expressing function of BR S35C. (c), (d) Displacement of $\theta$ ($\Delta {\theta }_{570}$ and $\Delta {\theta }_{400}$) of observed diffracted spots with $\sim 570\text{−}\mathrm{nm}$ and $\sim 400\text{−}\mathrm{nm}$ lights.

Figure 3

Curves of mean-square displacement $\left(\Delta {\theta }^2\right)$ of nanocrystals as a function of time interval $\Delta t$ among three regions for analyses of Brownian motions. Two insets show Figs. 2c, 2d. Six symbols represent the experimental results for 70 diffracted spots, while solid curves show the theoretical curve fittings.

Figure 4

Positional change of BR S35C by light irradiation $(\sim 570\mathrm{nm})$. The BR structure is the BR state (silver, PDB ID: 1C8R) superimposed onto intermediate $M$ (black, PDB ID: 1C8R). The solid arrow shows the positional change of BR S35C between the two states with the 131st amino acid as the center of rotation.