SP2271: Introduction to Scientific Literature - Assignment 3

This post is meant to showcase my work for the Assignment in my SP2271 class, which was held in high regard by the professors.

Assignment 3: Write a summary of the paper of your choice (in 400 words) and your own scientific commentary on the same paper (in 600 words).

Original paper of my choice: read it here.

Note: Wix.com does not allow me to use subscripts in this post, so please pardon the wrong formatting of F1-ATPase (the number 1 should be in subscript).

Summary:

The fluctuation theorem (FT), which deals with the probability of a system moving by different forces, has previously been applied to study small-scale biological systems, but not biomolecular motors. Torque calculation in biomolecular motors was generally performed with fluid dynamics, which studies the effect of different forces on the flow of fluid, but this method might provide inaccurate results. Hayashi et al.1 (2010) have hypothesised that FT can accurately calculate the torque of the biomolecular motor protein F1-adenosine triphosphatase (F1-ATPase). This research can potentially extend the usage of FT as a torque estimation technique in other biomolecular motors.

To achieve improved visualisation of the rotation of F1-ATPase, the paper probed it with two kinds of beads: an irregular-shaped magnetic bead or a duplex of spherical polystyrene ones. A camcorder was then employed to capture the angular position of the probe at different recording rates. After plotting the rotational angles ( ) against time, the 120º stepping of F1-ATPase was observed. This observation aligns well with current knowledge.

Torque measurement was based on two ideas: 1) assessing the speed at which the angular position of F1-ATPase changes and 2) evaluating the effects of random forces, or thermal noise, on the system. FT was employed to combine these two ideas, which can derive the torque of F1-ATPase from the probability distribution of the change in at fixed time intervals.

When comparing results with the literature, which established that the torque generated by F1-ATPase should be close to 40pN nm, the paper found that the torques measured by FT were closer to that value than those by fluid dynamics, be it stepping or continuous rotation and even with different sets of beads. Further examination of an F1-ATPase, which was probed by a magnetic bead, found that the torque was similar even at large time intervals and varied recording rates (500fps – 3000fps). These findings suggest that torque calculation by FT is more reliable. Even for a mutant type of F1-ATPase, the torque calculated did not deviate much, even though the pause between each 120º step was longer. When FT was applied to another motor protein, V1-ATPase, the torque was less than that of F1-ATPase, which is also consistent with the literature.

In essence, the paper concludes that FT can provide a more accurate torque calculation of F1-ATPase than fluid dynamics and proposes the application of FT in studying other biomolecular motors.

397 words.

Commentary:

The paper has found a way to attain improved visualisation of the rotation of F1-ATPase through the rotation of the probe attached to it. The accurate observation of the angular position of the probe is essential to the calculation of the torque2. Whilst previous studies predominantly used an actin filament to probe F1-ATPase3–5, this paper used spherical and irregular-shaped beads instead. One could posit that these beads were employed because they might cause little interference with the rotation of F1-ATPase. The actin filament may have a long and rod-like shape, making it easy to scratch the glass surface6. Irregular-shaped and spherical beads, on the other hand, have a round shape, therefore not easily scratching the glass surface as actin filament does. The usage of these beads allowed for clearer visualisations of the stepwise rotation of F1-ATPase than in a previous study that used an actin filament as the probe3.

Although the appropriate use of spherical and irregular-shaped beads did help the paper observe the stepwise rotation of F1-ATPase, the visualisation could be further improved. In the paper, stepwise rotation was not observed at high ATP concentration (1mM). This could have happened because the sizes of the beads were so big that it could still cause interference with the rotation of F1-ATPase. In order to get more accurate observations of the stepwise rotation of F1-ATPase, gold nanoparticles (AuNPs) could be a good candidate for making the probe. AuNPs have high compatibility with many biomolecules and high precision in imaging under optical microscopes6,7. A recent study has applied AuNPs to probe a close relative of F1-ATPase, V1-ATPase, and obtained fair visualisations of stepwise rotations of V1-ATPase even at varied ATP concentrations (100nM to 30mM)8. This highly suggests that AuNPs can also provide similar accurate visualisation of stepwise rotations for F1-ATPase. However, it should be borne in mind that the paper did a good job of improving the current method and visualising the rotation of F1-ATPase, considering that AuNPs were not well-developed at the time the research was being conducted.

The experimental setups, together with the apt use of FT, have generally fulfilled the primary aim of the paper, albeit with a little inconclusiveness in the results of further experiments. When FT is applied to a mutant type of F1-ATPase, the torque measured was close to that of the wild-type. The mutant type of F1-ATPase had glutamic acid in its -subunits replaced by aspartic acid. Because the -subunits of F1-ATPase are known to be the sites where reactions occur9,10, mutations at these sites can greatly impair F1-ATPase. In an earlier study11, which also introduced the same mutation, the activity level of F1-ATPase was found to have significantly gone down. Even though the paper did mention that the pause between each 120º step was longer in the mutant type of F1-ATPase compared to the wild-type, the difference in the pause time should be made clearer. Future work could measure the activity level of the mutant F1-ATPase from the data on the torque and the intervals between each 120º step, thus verifying that FT can provide reliable torque measurement even for a mutant type of F1-ATPase.

Overall, whilst there remains minor ambiguity in the results obtained from further experiments, it does not detract from the paper accomplishing its fundamental aims. The paper has greatly contributed to the advancement of the field and laid the ground for future research, by the same and other authors12–15. Further studies employing AuNPs as the probes for F1-ATPase should be conducted to further establish the accuracy of FT in the torque calculation of biomolecular motors.

597 words.

References

(1) Hayashi, K.; Ueno, H.; Iino, R.; Noji, H. Fluctuation Theorem Applied to F 1 -ATPase. Phys. Rev. Lett. 2010, 104 (21), 218103. https://doi.org/10.1103/PhysRevLett.104.218103.

(2) Evans, D. J.; Searles, D. J. The Fluctuation Theorem. Adv. Phys. 2002, 51 (7), 1529–1585. https://doi.org/10.1080/00018730210155133.

(3) Yasuda, R.; Noji, H.; Kinosita, K.; Yoshida, M. F1-ATPase Is a Highly Efficient Molecular Motor That Rotates with Discrete 120° Steps. Cell 1998, 93 (7), 1117–1124. https://doi.org/10.1016/S0092-8674(00)81456-7.

(4) Sakaki, N.; Shimo-Kon, R.; Adachi, K.; Itoh, H.; Furuike, S.; Muneyuki, E.; Yoshida, M.; Kinosita, K. One Rotary Mechanism for F1-ATPase over ATP Concentrations from Millimolar down to Nanomolar. Biophys. J. 2005, 88(3), 2047–2056. https://doi.org/10.1529/biophysj.104.054668.

(5) Nishizaka, T.; Mizutani, K.; Masaike, T. Single-Molecule Observation of Rotation of F1-ATPase through Microbeads. Methods Mol. Biol. Clifton NJ 2007, 392, 171–181. https://doi.org/10.1007/978-1-59745-490-2_12.

(6) Hu, X.; Zhang, Y.; Ding, T.; Liu, J.; Zhao, H. Multifunctional Gold Nanoparticles: A Novel Nanomaterial for Various Medical Applications and Biological Activities. Front. Bioeng. Biotechnol. 2020, 8, 990. https://doi.org/10.3389/fbioe.2020.00990.

(7) York, J.; Spetzler, D.; Hornung, T.; Ishmukhametov, R.; Martin, J.; Frasch, W. D. Abundance of Escherichia Coli F1-ATPase Molecules Observed to Rotate via Single-Molecule Microscopy with Gold Nanorod Probes. J. Bioenerg. Biomembr. 2007, 39 (5–6), 435–439. https://doi.org/10.1007/s10863-007-9114-x.

(8) Iida, T.; Minagawa, Y.; Ueno, H.; Kawai, F.; Murata, T.; Iino, R. Single-Molecule Analysis Reveals Rotational Substeps and Chemo-Mechanical Coupling Scheme of Enterococcus Hirae V1-ATPase. J. Biol. Chem. 2019, 294 (45), 17017–17030. https://doi.org/10.1074/jbc.RA119.008947.

(9) Leslie, A. G. W.; Walker, J. E. Structural Model of F 1 –ATPase and the Implications for Rotary Catalysis. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2000, 355 (1396), 465–471. https://doi.org/10.1098/rstb.2000.0588.

(10) Weber, J.; Senior, A. E. Catalytic Mechanism of F1-ATPase. Biochim. Biophys. Acta 1997, 1319 (1), 19–58. https://doi.org/10.1016/s0005-2728(96)00121-1.

(11) Amano, T.; Tozawa, K.; Yoshida, M.; Murakami, H. Spatial Precision of a Catalytic Carboxylate of F1-ATPase Beta Subunit Probed by Introducing Different Carboxylate-Containing Side Chains. FEBS Lett. 1994, 348 (1), 93–98. https://doi.org/10.1016/0014-5793(94)00588-5.

(12) Hasegawa, S.; Sagawa, T.; Ikeda, K.; Okada, Y.; Hayashi, K. Investigation of Multiple-Dynein Transport of Melanosomes by Non-Invasive Force Measurement Using Fluctuation Unit χ. Sci. Rep. 2019, 9 (1), 5099. https://doi.org/10.1038/s41598-019-41458-w.

(13) Hayashi, K. Application of the Fluctuation Theorem to Motor Proteins: From F1-ATPase to Axonal Cargo Transport by Kinesin and Dynein. Biophys. Rev. 2018, 10 (5), 1311–1321. https://doi.org/10.1007/s12551-018-0440-5.

(14) Hayashi, K.; Tanigawara, M.; Kishikawa, J. Measurements of the Driving Forces of Bio-Motors Using the Fluctuation Theorem. BIOPHYSICS 2012, 8 (0), 67–72. https://doi.org/10.2142/biophysics.8.67.

(15) Seifert, U. Stochastic Thermodynamics, Fluctuation Theorems and Molecular Machines. Rep. Prog. Phys. 2012,75 (12), 126001. https://doi.org/10.1088/0034-4885/75/12/126001.

Acknowledgement:

Thank you Prof. Yuan Zhe and Prof. Dara for helping me in the process of writing and polishing my writing.