Dude – this water’s so heavy. Is it deuterated? Deuterated water may be heavy but it isn’t “hot” which makes it helpful for seeing if protein regions are “open” or not. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is kinda like giving wetsuit-wearing proteins a bath and seeing where they get wet. You bathe them in heavy water and exposed unstructured regions of the protein get heavier, whereas the structured regions are hidden under the wetsuit so they stay dry.
I spent much of today finishing looking at data from this cool technique you can use to study protein shape & dynamics. Since I put so much time into learning it, figured I’d share what I learned in case anyone else was interested. First an overview then some more detail. So here’s the gist…
Deuterium (D) is a version of hydrogen which has 1 more neutron than “normal hydrogen” so it’s heavier -> when you bathe your protein in D₂O the protein can swap out H for D and this makes the protein heavier. And if you cut the protein up into pieces and weigh those pieces individually you can see where swapping occurred and didn’t occur, telling you about how accessible &/or structured those regions are. It’s often used to see if regions become more or less swappable under different conditions or after adding a binding partner.
If H is held tightly, it won’t get swapped, so it will stay “light.” But if H is in a solvent accessible region and it’s not tied up, it will get swapped out with the heavier version, deuterium. So when you then cut the protein up into pieces, the piece that was accessible will be heavier
The “weighing” is done by mass spectrometry or “mass spec.” It’s a technique that can be used to identify proteins and identify modifications to proteins – all based on how heavy and charged they (or at least parts of them) are. Not going into the technical stuff, the principal is that you use endoproteases (protein scissors) to cut up proteins into little pieces, then you charge those pieces – turn them into ions using electrospray – measure the weight of those pieces and, because different protein letters weigh different amounts, you can figure out what letters are in each piece and then match that up to the letters in protein sequences in a big database.
It’s kinda like “reverse-redacting” – you know the full text and you’re trying to see what parts of that text are covered (and covered in the sense that those letters were detected – not blacked out :P) mass-spec results come out as a series of peaks on an m/z graph, where m’s mass and z’s charge. Increased accessibility leads to increased deuteration leads to increased mass leads to rightward shift. Decreased accessibility leads to decreased deuteration leads to decreased mass leads to leftward shift
Different protein letters have different masses because they’re made up of different combinations of elements. All protein letters have a generic backbone (although proline’s is slightly different since it’ side chain kinda curves back to hog the N). But they have different side chains, which have different numbers and arrangements of atoms of carbon, oxygen, nitrogen, and/or sulfur. One thing they all have – hydrogen.
Hydrogen’s often “ignored” – sometimes it’s not even drawn in, its presence is just implied. Because it’s not very reactive. And speaking of activeness – hydrogen has heavy form that are NOT radioactive.
Atoms are made up of protons (+ charged), electrons (- charged), & neutrons. Different elements are defined by how many protons they have, but the number of electrons & neutrons is more flexible. http://bit.ly/2G01Kpg
The electrons get a lot more attention in biochemistry because they’re negatively charged and charge makes molecules want to do things like go towards or flee from other molecules. Electrons also get more attention because they’re the part of atoms that atoms share to form covalent bonds.
Neutrons, on the other hand, are neutral, and they’re in the nucleus, too far away to interact with other atoms. So normally we don’t think about them much they’re just kinda there in the background.
But the number of neutrons can vary without changing the identity of the atom, and we call these different version “isotopes.” So an atom with 1 proton is always hydrogen no matter how many electrons or neutrons it has. Of course, atoms can only hold a certain number of these. When atoms have more neutrons than they can handle, they’re radioactive & can decay to a less neutron-y state, letting of radiation in the process. And we can take advantage of this to radiolabel things like RNA to track it. more here: http://bit.ly/2VtYSG7
- add an electron and you get a hydride ion (OH⁻)
- remove an electron and you get a proton (H⁺), which normally hangs out with water as a hydronium ion (H₃O⁺)
- add a neutron and you get deuterium
- add two neutrons & you get tritium, which IS radioactive
Unlike radiolabeling, where we use radioactive isotopes, deuterium isn’t radioactive – it’s stable, just “different” from normal H. So deuterated water is heavy but not “hot” (slang for radioactive)
I remember in biochemistry & chemistry classes H’s would just seem to come & go out of nowhere in equations & mechanisms and it drove me crazy. But turns out hydrogen really does come and go quite readily – and frequently – if it’s attached to the “right things” – hydrogen is constantly being exchanged and we can take advantage of this to see where exchange is occurring and more significantly where it is NOT occurring
Water can exist as H₂O or H⁺ and OH⁻ and that H⁺ usually grabs on to another H₂O to give you H₃O⁺ (hydronium ion). So you have an OH⁻ able to take an H & H₂O & H₃O⁺ willing to give an H. They can give and take from each other (other water molecules) or they can give and take H’s from other things.
Same goes for deuterated water – it acts the same as normal water because the other molecules “ignore the neutrons” as well. So D₂O gives you D⁺ and DO⁻. And that DO⁻ can pull off the normal H, allowing it to get swapped out. But the DO⁻ has to find it, so it has to be solvent-accessible, and “unoccupied” and in order for us to be able to detect it it can’t be sooo swappable that it swaps back when we do the post-labeling stuff, which uses normal water.
Proteins have a lot of hydrogens, and here are several places you’ll find them. Most of them are attached to carbons, and these H don’t like to leave without a really good reason to – those are unlikely to just swap out for a hydrogen from the water. So the exchange rates for H in C-H bonds are too small to measure.
The H’s in side chain functional groups, like those in hydroxyl (-OH) and carboxyl (-COOH) groups have the opposite problem. They swap out so rapidly that when you quench the reaction in a normal water-based solution, they swap back to the light form, leaving no evidence that any change occurred in between. It’s like when you take them out of the bath, some regions dry off before you even knew they were wet.
But all hope’s not lost – there’s another place that you find H’s in proteins – in the amide (-(C=O)-NH-) functional groups in the generic backbone (aka backbone hydrogens). All the letters have it except for proline, whose side chain “loops back” to bind the N so the N doesn’t have electrons to share with the H. Some letters also have exchangeable H’s in N-H’s in their side chains as well (e.g. lysine and arginine).
The H’s in the N-H backbone bonds are exchangeable at a measurable rate – if they’re accessible that is. A lot of the time these H’s are tied up in hydrogen bonds with other atoms. In fact, a lot of protein structure comes from these H’s H-bonding to the carbonyl (C=O) oxygens of the backbones of other letters in other parts of the proteins. Such backbone-backbone interactions give the protein its “secondary structure” – things like alpha helices and beta strands.
H-bonds are not covalent (there’s no electron cloud merging) so they’re not as strong as the covalent bonds that actually give the protein it’s primary structure (connect the letters in linear fashion). But they add up to really glue the protein together. So in highly structured regions of the protein, those H’s won’t be available for swapping. Though if you wait long enough those bonds can break and reform as the protein “breathes” and this offers a chance to sneak in.
And speaking of time, what’s normally done is you deuterate for several different lengths of time – the longer it takes for an H to get swapped, the harder it is to find and/or the more tied-up it is. To stop it you “take away the DO- by adding acid, which neutralizes the DO- and lower the temperature – depriving molecules have of the energy needed to do all that swapping
Some things you can do with HDX-MS:
at the large scale – global HDX measures mass of the whole protein (no cutting it up first). you can do things like compare w/& without binding partner (ligand) -> tells you about overall binding (does it bind or not) under different conditions (e.g. at low pH, high pH, low salt, high salt, etc.) and/or w/different introduced protein mutations (e.g. if you think a residue is important for binding & you change that residue to a different letter, will you still get binding)
at the finer scale – “local HDX-MS” (with cutting*) – look at changes in specific regions of the protein. *Instead of cutting with enzymes (the bottom-up approach) you can break it in the gas phase in the “top-down” approach – instead of cutting the protein in lots of places it just cuts it in 1 place giving you 2 big pieces. But “each” of those pieces is cut somewhere different so you get different sized pieces and then you can compare their weights
link to a good review paper: https://www.nature.com/articles/s41592-019-0459-y
link to the paper I’m going to present: https://link.springer.com/article/10.1007/s13361-017-1830-9#Bib1
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0