Directed Evolution

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Directed Evolution

Postby stcordova » Fri Oct 05, 2018 11:01 am

as9/CAS13a/b/d for example are also proteins with pretty weird domains, the directed evolutions of which generated some truly crazy and weird structures not found elsewhere (doesn't mean nature wouldn't have evolved them someday though). Cool thing about them! The flexibility of their "variable" loop allowed for many varied directed evolutions off of them that were all unique.

Directed evolution of sa/spCas9 was able to generate approximation with homologs from one into the other. sa/sp are actually very similar, with one simply being the other but with 3 additional modules that evolved separately. Directed evolution of one was able get close to one from the other without explicitly trying to. Generation of xCas9, eSpCas9, etc. Cas9 has a very crazy thing it does relying on RNA-RNA, DNA-RNA, Protein-DNA, Protein-RNA interactions to accomplish its task and has a spiraling conformational change that occurs in wildtype when it finds its substrate as a result of reacting to this vortex of forces. This massive conformational change means that its evolution can generate weird new structures if any step along the way is altered.

ADAMTS family members are disintegrin metalloproteases that cut cell membranes to allow for motility. All of the members are different but generated through duplication events. Snake venom is one such member with a novel domain change that causes it to behave like a toxin through this method. Directed evolution of ADAM members then has the potential to compete against snake venom through a new domain being generated.

This sort of things happens often! Two randomized evolution experiments I did generated the same kinds of mutations despite being kept separate and done using different protocols- convergent evolution is a highly repeatable phenomenon.

I think you have this idea of "structure" being super rigid in your mind. Newsflash- novel structures are trivial to evolve because proteins are way more flexible than you think. For example. Serpinopathies are a whole subset of disease that occur because serpins, a class of proteins under high stress, misfold into a weird conformation with a single mutation. Boom, new structure. We have chaperone proteins because novel structures arise too easily.

Thank you for the informative data! That was the most informative thing I've heard in this sub, ever.
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Re: Directed Evolution

Postby stcordova » Fri Oct 05, 2018 8:10 pm

how many residue changes

Slaymaker et al 2016 covers the structure of cas9, which is a good reference.

There are quite a few residues which interact with RNA/DNA, with a total of 8 key contact points spread across 2 domains.

As for overall domain structure, there is a range between 1000-1350AA for the size of the proteins across species. The three subdomains necessary to differentiate them would be among those 350. I would venture to say ~100 mutations are necessary to reach an approximation. Some these don’t have to be AA changes though. Silent mutations can also change protein structure through codon optimization- slower codons can cause the kinematics of synthesis to change.

Mutation can occur through sequence duplication of a variable loop or through subunits, among other things.

How many residues converged

Not simply residues but also silent mutant convergence due to codon optimization.

Around 12-15 per round of evolution, depending on how confident we were whether the mutation was noise or a actually selected for.
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