rosie4loop Lv 1
(Edited for clarity and fix typos)
Always happy to see a plan before the start of a project, this is useful to estimate the resources required and the skill you need to equip. It is good to have a motivation to start a project. Indeed, drafting a research plan and search for the tool you would like to use is a good practice for any researchers.
So I had some fun trying to comment on it during a break, imagining this is an idea from researchers who work in other fields.
Let's break it down into different aspects to see if it is good to execute the current plan.
I'm trying to create an enzyme to fix a mutated protein into its original shape and function.
(1) Science question:
- Mutants M1, M2, M3… of a protein (lets call it P) lost its function due to mutation
-
Key assumption here:
- mutants M1, M2, M3… no longer function because of the changed shape upon mutation
- which means:
- if protein P is an enzyme, the mutated sites are not directly involved in the reaction
- if protein P is a kind of regulators that inhibit or activate other proteins, or if P is a signaling molecule that affect other downstream processes, after correcting the shape it can still bind its binding partner
- if protein P is a structural protein, the function can be restored by holding the shape with something else
(2) Main project objective:
- Restore the function of protein P from M1, M2, M3…
- by designing a new enzyme (lets call it E)
- E turns the shape of M123 back into P for its function
(3) What you need according to your statement:
- The correct shape of wildtype P
- Sequence of M123… and build their structure from the correct shape of P
- "Wrong" structure of M1, M2, M3…
- Enzyme E, assuming you need to design it, that restore the shape of M123 into the shape of P
- E need to bind the "wrong" structure of M1, M2, M3 to initiate the whole process (!!)
- After binding to the "wrong" M1, M2, M3… E need to force them back into the shape of P. (this is very challenging to investigate or to design, that E need to induce a change upon binding. May need energy)
- For an initial design of a small project maybe ok to use only one form of mutant (?)
- Metrices to decide whether the designed enzyme E is good enough for your purpose
- Experimental validation
(4) Intermediate goals you need to achieve to get the items in (3):
- get the structure of P (if exists)
- get the strcuture of M1, M2, M3…
- build the "corrected" fold of M1, M2, M3
- design the enzyme E
- evaluate different designs of enzyme E.
- (likely out of the scope of the project) model the process of E + M (wrong) -> E + M (correct)
(5) Comments and unanswered questions on each proposed step
I will collect the amino acid sequence of each mutated protein,
- First of all, double-check if the key assumption is valid that "P -> M1, M2, M3… mutants lost their functions because of conformational change upon mutation"
- Is there any known stucture(s) of the mutated proteins? If no, would it be important to model their structures first?
- Which of the following you would like to target? Is it the structure of the "wrong" shape of M1, M2, M3…? Or the structure of the "corrected" M1, M2, M3…?
and one at a time run foldit with it creating a new enzyme strand that will alter the mutated protein to its correct shape. (I understand that is not built in to Foldit, so I will create my own system using Python and the Script window provided in-game)
This can be breakdown into multiple steps (if I get it right):
- design the backbone of the enzyme E that bind M1, M2, M3…
- optimize the sequence of E to produce the fold you want
- predict the interaction between E and M1, M2, M3…
- predict if E interacts with the "wrong" structure of protein
- predict if E changes the shape of M1, M2, M3… upon binding
- predict whether the corrected structure of M123 can serve their fuction
(1)-(3) may be possible to do after you decide the target, but (4)-(6) are difficult, that Foldit may not be the ideal tool for it even if you can do exhaustive sampling.
Also how can you decide that the enzyme can alter the mutated protein to the correct shape? This kind of changes are complicated. It's already difficult to design a binder, even harder to design a machine that works after binding. (I'm curious if you know a tool that do it, let me know what it is!)
Afterwards, I will manually add in blocking structures to avoid the enzyme from collapsing.
- If you design a folded enzyme, why you need a blocking structure to avoid it from collapsing?
- Does it mean you just design a certain motif that "hold the shape", but without a stable structure? So the blocking structure is also a protein?
I will check my solution by having the enzyme in contact of every protein type it may encounter individually from all angles.
- I am not sure about the purpose of this step, or if I have misunderstood something. Why you need to bind it from all angles?
- If you design the enzyme to target a structure, or if protein P is known to bind something, you should know where you want it to bind already. If the interface is unknown it'd be difficult to evaluate.
- How to decide the contact from a certain angle is important or useful?
- Prediction of protein-protein interaction on without a known interface surface is challenging (actually still challenging even if the interface is known). How do you decide this binding mode is relevent?
(6) Other comments
- I don't know the scale or the level of this project, is it a project in class? Or a honor project for final year students? Or it is a personal project solely for personal interest? How much time you will have to finish it?
- If it is just a class project, have an rough prediction of the structure of mutants and discuss on how the mutation affects the functional sites may take a long time already.
- If you decide to use either the "correct" or the "wrong" fold of M1, M2, M3… as your binding target of an initial design
- need to tell the reason of using this conformation as the target
- lets say at this stage, designing a binder could be good enough within the time limit. So you have the "correct" shape of mutants and try to bind someting with it to stabilize.
- If you use Foldit, you need the PDB of "corrected" M1, M2, M3… as the taget, and a dummy chain for binder design. Like those binder design begineer puzzles in Foldit.
- Manually design a binder can be challenging and time-consuming (ask the rest of Foldit community). If you don't have enough time for that, you may try another tool "RFdiffusion" which is also related to Rosetta like Foldit. You may try it for free from Google colab (https://colab.research.google.com/github/sokrypton/ColabDesign/blob/v1.1.1/rf/examples/diffusion.ipynb). That tool let AI do the design instead of human.
- Even if you have a design of E that binds one form of M123, it's difficult to check if it could turn them back into a correct shape.
- Likely out of the scope of your project
- If more time is allowed and you have the resources and skill, I'd do some simulation of WT and mutants to investigate the stability, conformation changes (need to use other methods, not Foldit. But this is more of my personal intesest to know what happens.)
- There are some tools that predict the change in activity and thermodynamical properites upon mutation using AI. That sometimes also tell the reason from the structural perspective.
- model the process of E + M (wrong) -> E + M (correct) is challenging even for scientists. There are too many variables there, require many (computational) experiments. Also need to breakdown the project into smaller parts to do it.
(7) Final Remarks
Don't just rely on "random people on internet who claim they know something" (like the person who wrote these comments).
Read papers from peer-reviewed journals on the topic you are interested in, so you know the current trend, and how scientists design experiments to do something similar to what you want to do. Also disuss with people who know you better, like your mentors or instructors. So you know more on what kind of knowledge you need to learn and what kind of skill you need to have, until you get what you desired.
And good luck on your project!
