Team:NYMU-Taipei/optomagnetic-design

It is known that transmembrane (TM) proteins usually have high propensities between the inter-helices. Mms13 possesses two tightly-interacting transmembrane helices present as the major obstacle within our optomagnetic design, since theoretically, BRET will be an constitutive phenomena, leading to uninhibited excitation / unregulated inhibition of target neurons. However, the anticipated model we proposed rooted heavily in induced BRET with conformational wobbling within mms13, functioning as switches for neurons shifting back and forth between "ON" and "OFF" states.

We found inspiration from a paper associated with the bimolecular fluorescence complementation (BiFC) method. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein. When those fragments are brought in proximity to each other by an interaction between proteins, they would fuse into one functional fluorescent protein (Tom K. Kerppola., 2008). We came up with the idea by mixing the BiFC method and BRET phenomenon into the BiFC-based BRET design. (See Figure 2)

In our BiFC-based BRET design, we anchored a half of a YFP fragment (with YFP C-terminus) on the inter-helical inhibitor (the CHAMP peptide design). By using the inhibitor, we can affect the tight interaction between Mms13’s two helices, and the constant light induced form BRET phenomenon can also be blocked. The rest of the design is to get the other half of the YFP (with YFP N-terminus) anchored on the N-terminus of Mms13 and anchored the r-Luciferase on C-terminus of Mms13. The result is that when there is no magnetic force applied to Mms13, two helices of Mms13 will have further inter-helical distance due to the inhibitor (CHAMP design) and the BRET phenomenon will not perform due to the lack of close proximity between two helices. When we apply magnetic force to Mms13, the helices of the transmembrane protein will be pulled together and will induced the BRET phenomenon to emit fluorescence.

The CHAMP Design

CHAMP, the computed helical anti-membrane protein, is designed by one of the computational and genetic methods available to engineer antibody-like molecules that target the water-soluble regions of tansmembrane (TM) proteins (Hang Yin, et al., 2007).

In the project, we want to use this designed peptide, CHAMP sequence, to inhibit the tight interaction between two helices of membrane protein Mms13 so that we can successfully use mechanical force to change the conformation of Mms13 to induce the BiFC-based BRET phenomenon. The details of the CHAMP design is shown in protein modelling.

Our Constructs

We have six constructs for our design including both positive and negative controls.

Fig. 3: The main design construct. Ori is the origin of replication of AMB-1. Pmsp1 is the promoter for AMB-1.

Fig. 4: A construct without YC on the CHAMP is our negative control in our design. It would not light up no matter there is magnetic field applied or not.

Fig. 5:. We design this construct with the whole YFP on the N-terminus of Mms13 and the CHAMP design to induce the BRET phenomenon. We suppose that with the CHMAP design, the construct will light up only when magnetic force is applied.

Fig. 6: This design is constructed with the whole YFP on the N-terminus of Mms13 but “without” the CHAMP design. The construct should be a positive control with constant light no matter whether force is applied or not.

Fig. 7: The design construct is similar with construct in Fig. 5. Why we construct this design with the whole YFP on the C-terminus of the CHAMP design instead of Mms13’s N-terminus is that we try to avoid the interferences of DNA transformation with sequences added in front of the natural construct. The construct is supposed to light up when force pull the Mms13 and turn off if no magnetic field is applied.

Fig. 8: In this last construct, only r-luciferase anchored on Mms13’s C-terminus. It will light up in yellow light (with 580 nm wavelength) due to the luciferase reaction, instead of the green light induced from BRET phenomenon (540 nm wavelength).

Modeling For Optomagnetic Design

Though we have already done the design part of our project, we may be challenged by whether it can function in reality or not. We consulted with Professor Chun-Min Lo who has a professional background in biomechanics. One of his research interests is to apply magnetic force on the super magnetic particles contact on the cell membrane and measure the cell’s morphology and biomechanics to study the growth of cells. After we told him about our project and our final goal to achieve, he showed interest and was willing to help us in calculating and modeling our optomagnetic design and he also generously supplied electromagnets, a type of magnet in which the magnetic field is produced by the flow of electric current, in his lab. We then modeled and calculated the force and magnetic field applied to the magnetic bacteria by using Professor Chun-Min Lo’s devices. (CM Lo, et al., 1998)

Fig. 4: Magnetic bead force application. A schematic of the experimental setup, with the electromagnet device shown in cross section. (Joseph N. Fass, et al., 2003) The difference between our project and the study is that there is no need to bind the magnetic particles on cell membrane, since AMB-1 has its own magnetic beads (magnetosomes) and AMB-1 can be simply put on the medium they used to live and grow without adding other chemicals.

Activation-Required Magnetic Force

We calculated the force per unit volume of magnetosome by electronic balance, and also from the measured field of the permanent magnet. (Glogauer and Ferrier, 1998) The magnitude of the magnetic force per unit volume of iron oxide beads would be

Where M is the magnetization of the beads and δBz/δz is the gradient of the magnetic field along the normal line to the center of the pole face.

The magnetosome crystal is known to have high chemical purity so its magnetization can reach to the saturation limit for 3*10⁵ A/m. The average diameter of the magnetosome is 46 nm in magnetic bacteria, so the volume per bead will be 4.077*10^-22 m³. By calculating the data in the paper, we discovered that the cell can have very large scale of deformation when only around 2 nN is applied. With just 2 nN force, we can that track that cell and we can then trace back to the gradient of the magnetic field we should apply to the bacteria.

The average number of magnetosomes in magnetic bacteria is 20 particles. We should apply 2 nN/20=0.1 nN to one magnetosome. Dividing this by the total magnetosome volume (4.077*10^-22 m³), there would be 0.1 nN / (4.077*10^-22 m³)≈2.453*10¹¹ N/m³ for force per volume. The gradient we need can be measured by dividing the force applied per volume to the saturated magnetization, which is: (2.453*10¹¹ N/m³)/(3*10⁵ A/m)≈8.176*10⁵ T/m.

However, we certainly do not need the high gradient of up to 8.176*10⁵ T/m. Because the need to pull the membrane and change the conformation of proteins would not require that much force. If the devices we apply now would not strong enough to change Mms13’s conformation, there is still nothing to worry about because we can add more turns per unit length of electromagnet to increase the magnetic gradient and thus induce stronger force to fulfill our goal.

Fig. 6: A magnetic trap for high-force cell probing is typically composed of an electromagnet with a sharp tip on a micromanipulator. (Hayden Huang, et al., 2004) If the force is not strong enough to change Mms13’s conformation we can add more turns per unit length of electromagnet to increase the magnetic gradient and thus induce stronger force to fulfill our goal.

Fig. 5: Neurite initiation and elongation in response to applied force of only 450 pN. (Joseph N. Fass, et al., 2003)

Fig. 7: Measurements of magnetic devices-Electromagnet Gausses at Measured Distances by AZeer Enterprises, LLC

References

• Johan Bacart, Caroline Corbel, Ralf Jockers, Stéphane Bach, Cyril Couturier Dr. (2008) The BRET technology and its application to screening assays. Biotechnology Journal Volume 3, Issue 3: 311–324

• Kevin D G Pfleger, Karin A Eidne. (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nature Methods 3:165 - 174

• Tom K. Kerppola. (2008) Bimolecular Fluorescence Complementation (BiFC) Analysis as a Probe of Protein Interactions in Living Cells. Annu Rev Biophys 37: 465–487.

• Hang Yin, Joanna S. Slusky, Bryan W. Berger, Robin S. Walters, Gaston Vilaire, Rustem I. Litvinov, James D. Lear, Gregory A. Caputo, Joel S. Bennett, William F. DeGrado (2007). Supporting Online Material for Computational Design of Peptides That Target Transmembrane Helices. Science 315, 1817.

• J. S. Slusky, H. Yin and W. F. DeGrado. (2009) Understanding Membrane Proteins. How to Design Inhibitors of Transmembrane Protein—Protein Interactions. PROTEIN ENGINEERING Nucleic Acids and Molecular Biology 22, 315-337.

• Michael Glogauer and J. Ferrier. (1998) A new method for application of force to cells via ferric oxide beads. PFLÜGERS ARCHIV EUROPEAN JOURNAL OF PHYSIOLOGY 435, 2:320-327.

• Chun-Min Lo, Michael Glogauer, Marisa Rossi and J. Ferrier. (1998) Cell-substrate separation: effect of applied force and temperature. EUROPEAN BIOPHYSICS JOURNAL 27, 1: 9-17

• Hayden Huang, Roger D. Kamm, and Richard T. Lee. (2004) Cell mechanics and mechanotransduction: pathways, probes, and physiology. Am J Physiol Cell Physiol 287: C1–C11.

• Joseph N. Fass and David J. Odde. (2003) Tensile Force-Dependent Neurite Elicitation via Anti-b1 Integrin Antibody-Coated Magnetic Beads. Biophysical Journal Volume 85:623–636 623.

• Hayden Huang, Jeremy Sylvan, Maxine Jonas, Rita Barresi, Peter T. C. So, Kevin P. Campbell, and Richard T. Lee. (2005) Cell stiffness and receptors: evidence for cytoskeletal subnetworks. Am J Physiol Cell Physiol 288: C72–C80.

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