Introduction
Bacteriorhodopsin (BR) is a protein found in halobacteria that belongs to the Archaea family. BR serves as a proton pump that captures light energy and uses it to move protons across the membrane. The proton (pH) gradient produced is then utilized to produce adenosine triphosphate (ATP), which is the chemical energy source used for various protein processes within a cell (Figure 1).
Figure 1. Proton gradient used to generate ATP
Image Credit: Nanosurf
In native membranes, BR is present in large quantities and arranges itself in a crystalline manner. The molecules are arranged as trimers and both sides (extracellular and cytoplasmic) can be imaged using atomic force microscopy (AFM), as shown in Figure 2.
Figure 2. Extracellular side and cytoplasmic side imaged by AFM
Image Credit: Nanosurf
Imaging Bacteriorhodopsin with Nanosurf FlexAFM
BR is measured in buffer solution using Nanosurf’s FlexAFM system and C3000 controller. With this technique, two membrane patches containing BR are used; the right patch faces the cytoplasmic side up, while the left patch faces the extracellular side up. The difference in height is caused by unequal electrostatic interactions between the surface and the tip. Finally, data processing is performed using Nanosurf Analysis software (Figure 3).
Figure 3. Imaging BR with FlexAFM
Image Credit: Nanosurf
High Resolution Imaging of the Cytoplasmic Side of Bacteriorhodopsin
The images are unfiltered data with linear background correction. Figure 4 illustrates a correlation average with three trimers emphasized by rounded triangles. The image was then determined in static mode using the following:
- Buffer: 25mM MgCl2, 150mM NaCl, and 50mM Tris pH 7.6
- C3000 controller
- FlexAFM with 10µm scanner range
- Cantilever: 0.1N/m (Uniqprobe, qp-CONT, Nanosensors)
To calculate cross correlation, IPLT software was used.
Figure 4. High resolution imaging of the cytoplasmic side of BR
Image Credit: Nanosurf
Estimation of Resolution
For two-dimensional crystals, the resolution of the captured data can be calculated from the 2D power spectrum. In Figure 5, diffraction spots go further than 1nm lateral resolution (circle).
Figure 5. Recorded in buffer with: FlexAFM V3 with 10µm scanner range and C3000 controller
Image Credit: Nanosurf
Single Molecule Force Spectroscopy
Single molecule force spectroscopy is generally performed using soft cantilevers ≤ 0.2N/m such as qp-CONT and nanosensors. The spring constant has to be measured, for example, from thermal noise spectrum using John Sader’s method (Figure 6).
Figure 6. Thermal noise analysis in C3000 controller.
Image Credit: Nanosurf
Unfolding Bacteriorhodopsin Using Single Molecule Force Spectroscopy
BR can be unfolded using single molecule force spectroscopy as follows:
- Protein can be stepwise unfolded when pulling from a terminus (Figure 7)
- Binding is unspecific between protein and tip
- Precise unfolding pathway differs between molecules, but major barriers exist such as the membrane being penetrated by the structure
- It is possible to determine the length of the unfolded part from worm-like chain (WLC) fits. Where a fitted contour length is equivalent to 88 amino acids (aa), an additional force is required to pull out the next part starting at aa 89 from the pulling side
Figure 7. Unfolding of protein step-by-step when pulling from a terminus
Image Credit: Nanosurf
Figure 8 shows a single curve on the left and overlay of multiple curves on the right. Here, unfolding pathways differ between molecules.
- WLC contour lengths: 88 aa, 148aa, 219aa
- Noise level: FRMS=7.98 pN
- Recorded in buffer solution with uniqprobe, qp-CONT, nanosensors, k=0.1N/m and FlexAFM with 10µm scanner range and C3000 controller
Figure 8. Single curve (left) and overlay of multiple curves (right)
Image Credit: Nanosurf
Conclusion
High resolution imaging of BR using single molecule force spectroscopy not only helps in calculating the length of the unfolded BR, but also helps determine its exact unfolding pathways and how such pathways differ between molecules. The use of tools such as the FlexAFM and C3000 controller makes it easy to measure BR in buffer solution.
About
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