SITE-DIRECTED MUTAGENESIS ⚡️ If protein letters you insert, can new powers you exert? 🤔 If letters you delete, can you better compete? Or maybe you’ll find that the substrate can’t bind? Having some doubt? How about you swap letters out? 👉 👉 STRUCTURE AND FUNCTION are intimately connected, as you can appreciate by thinking about a spoon versus a knife. The same holds true for biochemical “utensils” like proteins, and we can exploit this relationship to learn about function from structure and structure from function by making specific changes to specific parts using SITE-DIRECTED MUTAGENESIS, then testing to see if those changes affected the protein’s activity.
The 3D structure of a protein comes mainly from the sequence of its amino acid building blocks, which is specified by the DNA instructions in its gene. So if we change the gene, we can change the protein and potentially its functioning. More on this gene to protein connection here: http://bit.ly/2G01Kpg
Some proteins are like stand-alone utensils, in that they only do “1 thing” but many proteins are more like Swiss Army knives – they can do multiple things, and they split those duties up among various parts called DOMAINS.
By mutating different domains, you can find which ones are important for what. Like how you could dull a knife part without affecting the bottle opener part. So keep in mind that there could be changes happening to different functions that you just don’t know about because you aren’t looking for them! You can only detect changes in the thing you’re measuring.
Even within a domain, not all parts are equally important for the task you’re measuring. For example, you could make a hole in a spoon’s handle without affecting your ability to slurp your soup, but if you make a hole in the spoony part you’ll dribble all over yourself. By mutating different parts, you can find what parts are important for what.
We can make different kinds of mutations, like swapping some littors (substitutions), addddding new ones (insertions), or remvng some (deletions). Some notations we use to describe what we’ve done to it👇
🔹 For substitutions, we put the original letter first & what it’s changed to afterwards. So, say you changed the 91st amino acid from a glycine (G) to an alanine (A). You could write this as G91A.
🔹 And, to indicate taking out stretches of amino acids, we use the delta sign, Δ. So if we removed amino acids 80-100, we would write Δ80-100
We can also do things like mix and match different parts of proteins. For example, Frances Arnold won the 2018 Nobel Prize in Chemistry for research on directed evolution, but her lab also studies how to combine pieces of different proteins together to make “chimeras” with cool new functions. She uses knowledge of the structures of the “parent” molecules to know where are good places to cut & paste so that the parts remain functional
I think today’s topic is best covered through an example (how polynucleotide kinase phosphatase (PNKP) helps make broken DNA fixable) in figure form, so see you in the pics!
Note: If you want to play around with the PNPK structure in 3D, I generated the figure using a cool new tool from the NCI called iCn3D. Glover et. al. ( 10.1016/j.molcel.2005.02.012 ) did all the hard work crystallizing it and solving the structure, then they deposited that information into a repository called the Protein DataBase (PDB) so anyone can use it. Each thing that’s deposited gets a unique PDB ID, and this one is 1YJ5. It’s actually the mouse version of the protein and you may notice some missing regions – this is because flexible regions of proteins are “camera shy” because of how crystallography works (you need lots and lots of molecules to freeze in the exact same position) – lots more on how crystallography works starting here: http://bit.ly/2qlzlRS
But anyways, a cool thing about iCn3D is that it’s web-based and you can save URL links that allow you (or anyone else) to “pick up where you left off” – so go ahead and play around!https://icn3d.page.link/QpnSwRpJyEPEAvVW8
And check out PDB-101 for more great resources and information about where these models come from!