Thread by @bj_charles

🚨 Interested in single-molecule imaging approaches and/or pore forming proteins? Check out this awesome work from Conall, James and Till at UNSW. Buckle up... 🧵

#UNSW @SingMolSci @Mol_Machines #SingleMolecule #imaging @PratoPores @_DrCJM_ @m_dunstone

doi.org/10.1101/2021.1…

Hold up... if you haven't already done so - follow @Mol_Machines to keep up to date with their awesome single-molecule studies :)

https://twitter.com/Mol_Machines/status/1450754619764736002?s=20

In essence, the study uses #TIRF microscopy to extract #kinetic information, from movies like the one below, for the entire assembly pathway of the pore-forming protein, PFO. What might look like the night sky disappearing, is actually hundreds of independent pore-forming events.

This new approach to studying pore assembly in microfluidic reaction chambers reveals many mechanistic steps and does so with time resolution. I'm very grateful to be involved in this project. I learnt so many new things... What new insights were learnt?

In the past, MACPF/CDC were through to form pores via a three-step mechanism. Monomers bind to the membrane, oligomerise into prepore assemblies and then some trigger causes the prepore to transition into a pore rupturing the membrane.

#AFM studies (by @hoogenboom_lab) found that perforin and sulilysin could insert into the bilayer as arcs (in contrast to full prepores). In the case of perforin, these inserted arcs count recruit additional monomers and continue to grow into a full pore. nature.com/articles/nnano…

So, what new insights did the single-molecule TIRF study reveal? Since the method is single-molecule and time-resolved, individual processes in the assembly pathway can be disentangled and studied independently.

These include membrane binding, nucleation, oligomerisation, pore insertion, recruitment and even some other strange effects…

The 1st insight. Nucleation of oligomer growth appears to be at the level of a dimer. This is also the rate limiting step. PFO dimers are exceptionally stable and represent the committed step in pore formation. We suspect due to rearrangements in the MACPF/CDC domain.

The 2nd insight. Poration – the event of inserting into the membrane – is controlled by energetics. In the oligomeric state one can consider the monomer to have a lowered activation energy. At any time if a monomer overcomes the threshold the whole complexe proceeds to insert.

A consequence of this model is that the larger the oligomer, the higher the chance it will insert. i.e. insertion rate is dependent on arc length. This does away with the concept of a “trigger”, as oligomerisation is sufficient to affect the activation energy of a monomer.

The 3rd insight. The rate of poration is also concentration-dependent (i.e. [PFO])… how is this possible? One idea is that younger arcs (i.e. those which more recently gained a monomer) are in a higher energy state. Maybe due to conformational changes on the end of the arc?🤷‍♂️

Penultimate insight: in contrast to previous observations, PFO polymerisation was observed uninterrupted after pore formation. This result suggests PFO oligomers can continued to recruit monomers after insertion.

This result is unexpected, largely because this wasn't thought to be possible. We discussed at length the different possibilities, but this is still an open question for future work... 😇

Last insight, my personal favourite. The assembly pathway is governed by kinetics. Any given pathway is in principal possible and each conceivable intermediate can be sampled. But the likelihood of that pathway (based on kinetics) define the mechanism.

E.g. a strictly "prepore" pathway is far more likely at low temperature, conversely arc insertion and continual growth is far more likely at high concentration, etc...

Some other interesting points: Oligomerisation is irreversible, there is no equilibrium (detectable) of oligomers. It is a one-way street… implications for drug discovery programs, as non-equilibrium processes can not be inhibited in the same way as equilibrium processes.

In other words, pore-forming proteins have an “escape pathway” from drug molecules.

If you made it this far, well done! A huge thank you to Till, James and Conall for including me in their research. I had an absolute blast - I learnt a tonne. It's great fun to branch out of your comfort zone once in a while, I look forward to more to come.

Want to know more? Check out the equally intertesting study on the same topic from @markianwallace at King's College London. Unlike the UNSW study, Senior et al can measure diffusion coefficients and observe coalescence of arcs! Pretty neat biorxiv.org/content/10.110…

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