Today I His-tagged my His-smas tree… HISTIDINE (His, H) may be an amino *acid* but it can also be a base (or a “double” acid)! When it comes to protons (H⁺), it’s a game of give & take! And His’ metal-binding ability can be used to make nickel-linked little beads “give and take” “His-tagged” proteins to help you purify them in Immobilized Metal Affinity Chromatography (IMAC). 

blog form: 

It’s Day 14 of #20DaysOfAminoAcids – the bumbling biochemist’s version of an advent calendar. Amino acids are the building blocks of proteins. There are 20 (common) genetically-specified ones, each with a generic backbone with to allow for linking up through peptide bonds to form chains (polypeptides) that fold up into functional proteins, as well as unique side chains (aka “R groups” that stick off like charms from a charm bracelet). Each day I’m going to bring you the story of one of these “charms” – what we know about it and how we know about it, where it comes from, where it goes, and outstanding questions nobody knows. ⠀

More on amino acids in general here but the basic overview is:⠀

amino acids have generic “amino” (NH₃⁺/NH₂) & “carboxyl” (COOH/COO⁻) groups that let them link up together through peptide bonds (N links to C, H₂O lost, and the remaining “residual” parts are called residues). The reason for the “2 options” in parentheses is that these groups’ protonation state (how many protons (H⁺ ) they have) depends on the pH (which is a measure of how many free H⁺ are around to take).⠀

Those generic parts are attached to a central “alpha carbon” (Ca), which is also attached to one of 20 unique side chains (“R groups”) which have different properties (big, small, hydrophilic (water-loving), hydrophobic (water-avoided), etc.) & proteins have different combos of them, so the proteins have different properties. And we can get a better appreciation and understanding of proteins if we look at those letters. So, today let’s look at Histidine (His, H).

Histidine is AROMATIC, but unlike the other aromatic amino acids we’ve seen (tryptophan, tyrosine, & phenylalanine), His is + charged (at least sometimes) – let me explain, starting with what aromatic-ness means. Atoms (like individual C’s and H’s and O’s) are made up of smaller parts called subatomic particles; namely positively-charged protons and neutral neutrons which hang out in a dense central nucleus and negatively-charged electrons which whizz around them in an “electron cloud.” Atoms link up to form molecules by sharing pairs of electrons – you need 2 for a single bond & 4 for one of the shorter, stronger, double bonds. 

But what if you don’t quite have enough? You might want to join an electron commune! (otherwise know as resonance/electron delocalization/conjugation). Histidine’s a fan of this. Histidine is AROMATIC. That doesn’t mean it smells nice, it just means that it has a ring where, after they’ve “spent” 1 electron each on the bonds to their neighbors the atoms in the ring donate their “extra” into a communal shared stock. Those atoms that opt into this commune get to share, and this leads to electron delocalization above and below aromatic rings, kinda like a donut. 

His’ side chain has a 5-member ring and 2 of this ring’s corners (yeah, it’s more of a polygon than an actual ring…) are nitrogens (N) instead of the “usual” carbon (so we call it heterocyclic – hetero meaning different). These N’s are able to join the commune because they have a loan pair of e⁻ that can take “role” of double bonds in resonance structures in providing “extra” e⁻ 

One or both of this N’s may be bound to H. Under normal bodily (physiological) conditions, *at least* 1 of these N is protonated (bound to an H). If only 1’s protonated, we call this group IMIDAZOLE &, since the # of protons and electrons (being oppositely but equally charged) balance out, it’s neutral. But when the pH is low enough (i.e. there’s lots of free H⁺ around), the 2nd N will grab one too. And now you have more protons (+ charged) than electrons (- charged) and this makes for a ➕ charged (cationic) IMIDAZOLIUM ION

The ➕ charge in the imidazolium ion is shared between the 2 N’s. It might not look like it in drawings, but they’re basically equivalent – it’s just that if we only show a single “resonance structure,” where we draw only single & double bonds to show the “extremes,” it looks like one has to take on all the burden. We can draw resonance structures for His like the ones we drew for benzene & see this – but remember that these structures don’t actually exist – the real thing is somewhere in-between.

But in order to get that charged state, the pH has to be low enough. How low must you go? Here’s where things get interesting! pH is a measure of proton availability. It’s a negative log of the *inverse* of the H⁺ concentration ([H⁺]) which means that lower pH (more acidic) corresponds to higher H⁺ concentration and higher pH (more alkaline/basic corresponds to lower H⁺ concentration. The logging also makes it so that small differences in the pH correspond to much more dramatic differences in H⁺ concentration. 

The lower the pH, the more protons a molecule will collide with and each collision is a chance to grab on if they want to – some molecules want to, some don’t, some will if you go low enough. 

As you might remember from our discussion of acids (H⁺ donors) & bases (H⁺ acceptors) the strength of an acid (how willing it is to give up a H⁺) is described by its pKa value. When pH = pKa, 1/2 of the thing is  protonated; when pH < pKa, the protonated form is favored (since there’s an “excess” of protons) & vice versa (above the pKa there’s not much to take so you can think of it kinda donating a proton to the cause – though in reality it’s not doing it out of the Christmas spirit of giving, it’s just less likely to bind another one if it happens to let go). Imidazole’s 2nd N has a pKa ~6.5, which is close to biological pH (~7.4), so His can switch back & forth between protonated & deprotonated. 

note: I say “2nd” but “2nd” could be either since, as we saw w/the resonance structures, they’re basically the same, either can give up a H⁺ to give you 2 different “tautomers.” pKa is also context-dependent, so the placement of His residues within a protein can lead it to favor one or the other form. 

There are 3 amino acids that are commonly + charged at normal bodily (physiological) pH (~7.4), but the other 2 (arginine & lysine) have higher pKas, so they’re much less back-and-forthable in our bodies and are instead almost always protonated. The give-and-take-a-proton ability of His can be hugely helpful for enzymes (reaction speed-uppers) which can use it in their active sites (the pockets on the enzyme where reactions take place) in order to protonate and deprotonate reactants & reaction intermediates. His can also be used to facilitate reactions by stealing a proton from another amino acid to make that amino acid more reactive. 

Speaking of reactions, removing the carboxylate from His gives you HISTAMINE, which contributes to the symptoms of allergic reactions. That decarboxylation is done by an aptly named enzyme – histamine decarboxylase. Histidine can also get broken down by histidase (histidine ammonia lyase) and then other enzymes. Ultimately, if you choose to go down one of the metabolic path options, you can wind up with the amino acid glutamate, and you can use that to make glucose (blood sugar), so we call His glucogenic. 

N & NH can both participate in hydrogen-bonds (including w/water), so His is HYDROPHILIC and you can find it in water-exposed places on protons. Hopefully you can also find it in your food because it’s essential in the dietary sense meaning you need to get it “premade” in your food instead of making it yourself. 

Now let’s get back to that aromaticity – His’s aromaticity attracts ➕ charged metal ions. And this is really useful because metals are really useful – both in your body and in the lab. A lot of enzymes bind metals & use them as “helpers.” Why are metals so helpful? For one thing, they’re really big & multiple parts of the enzyme can latch onto them, so they can act as a sort of coordinating center to keep the enzyme’s structure together. 

Why so big? Metals have huge electron clouds – remember, those are the places where electrons whizz around their atomic nuclei containing the positive protons reigning them in. When metals bond to other metals in metallic bonds, they kinda just merge some of their clouds into a vast, communal electron sea. And when electrons move throughout the sea you have electric current, which is why metals are good electrical conductors.

But when metals bond to nonmetals, they often do so as COORDINATE COVALENT BONDS – these bonds form when one atom shares 2 electrons with a metal ion in the middle (in normal covalent bonds each atom shares one) – we call the result a COMPLEX and you can learn more about them here:

In order to complex with a metal, an atom has to have a lone pair of electrons they can share – like NITROGEN!!!!!. If a molecule has more than one atom with a pair to spare, they can “bite down” on the metal in multiple places -> POLYDENTATE. We call such multiple-toothed-biters CHELATORS

His’ N’s can act as electron pair donors in coordinate covalent bonds, so His is able to take a bite. And this can hold metals in place in the active site of enzymes, where those metals can give & take electrons to help promote reactions & stabilize awkward reaction intermediates. 

If you have a lot of His in a row, it’s like having a polydendate (many-biting) thing – one of those chelators. So if something has a string of His it can bind to metal tightly. And we can take advantage of this to help us purify proteins. 

If we want to study a protein, we usually need a lot of it. And we need it pure. So we express it recombinantly – this means we take the gene with instructions for the protein we want, recombine it with a piece of DNA that makes it easier to work with called a vector, then stick that vector into cells (often bacteria or insect cells) to make the protein for us. They’ll make it, but it’s up to us to get it out of the cells and purified.

So we want a way to make it easier to isolate just the protein we want and not any of the other stuff. A way we usually do this is through COLUMN CHROMATOGRAPHY. I flow a solution (MOBILE PHASE) containing my protein & contaminants I want to get rid of through a RESIN (lots of tiny beads) that’s held put in a column (SOLID or STATIONARY PHASE). This resin interacts w/different things differently. You want it interact w/your protein one way & the proteins you don’t want another way so that they get separated. Much more on this here:

We can use different types of resin to separate based on different properties – properties that are “inherently normal” to the protein – like size or charge or something we’ve added on through recombinant expression trickery. AFFINITY CHROMATOGRAPHY uses resin that recognizes something really specific – usually an affinity tag we’ve added onto the end of the protein (by putting the genetic instructions for it before or after the instructions for our gene in the plasmid). Because affinity chromatography is recognizing something “unnatural” and highly specific, it can (hopefully) remove most contaminating proteins – but not all.

A common affinity tag is a His tag, which is just 6 or 8 Histidines, which will bind to a Nickel (Ni) or Cobalt (Co) coated column (Immobilized Metal Affinity Chromatography; IMAC). Other proteins have Histidines too. But not that many in a row, so your protein will bind preferentially. It’ll hog the column and the other proteins will flow through.

Then you need something that will outcompete the His tag to get your protein off. Bring on the imidazole. It looks like His, so you can flood the column with imidazole to push the His-tagged proteins off. But before you flood it, you wash it with low levels of imidazole to remove non-specific binders that are just binding cuz they happen to have a lot of Hises.

So you go from 


resin:Ni:His-tagged protein

then resin:Ni:imidazole + His-tagged protein

In the beginning, when you’re loading your column, flowing through all that gunk, your protein is able to push off the imidazole because Imidazole mimics a single His so can only take a single bite whereas your tagged protein has a lot of them. When you are ready to push your protein off (once you’ve washed off all the other stuff) you can do it with imidazole, but you have to add a ton of it since your protein’s a better binder – you have to go with the “flood it out” strategy. more here:

Now for a bit of His-tory… If you’ve gotten tired of a bunch of these amino acids being first discovered in the milk protein casein, here’s a nice change – His was found in fish sperm. It was discovered simultaneously but independently in 1896 by Albrecht Kossel (who was investigating a weird thing in sperm) and Sven Gustaf Hedin (who was trying to figure out what was contaminating his arginine preps). Kossel did the naming, based on the Greek for “tissue”  

how does it measure up?

coded for by: CAU, CAC
systematic name: 2-Amino-3-(1H-imidazol-4-yl)propanoic acid
chemical formula: C6H9N3O2
molar mass: 155.157 g·mol−1

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉

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