What’s Tweety bird’s fave amino acid? tWiptophan! Tryptophan (Trp) is abbreviated W & you can remember this by saying it like Tweety! W’s one of my faves too & I’ll try to explain why w/o tripping up! You might have heard of it in the context of a different bird – yup, Tryptophan’s the source of the “turkey makes you tired” MYTH because Trp is a precursor to the chemical signaling molecules serotonin & melatonin that help regulate mood and sleepiness and stuff. Today, I’ll tell you why you should pardon the turkey – and why Trp helps us “see” our protein in UV.
blog form (refreshed from last December): http://bit.ly/tryptophanfan
It’s Day 9 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. ⠀
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 Tryptophan (Trp, W)
Like all molecules, Trp is made up of atoms (individual carbons, hydrogens, etc.). Atoms link up 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). Tryptophan’s a fan of this. Tryptophan is AROMATIC. That doesn’t mean it smells nice, it just means that it has rings and, in the rings, after they’ve “spent” 1 electron each on the bonds to their neighbors they 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.
Trp’s side chain’s a big, bulky INDOLE w/a methylene (CH₂) linker. INDOLE is BICYCLIC (2 linked rings). It has a 6 carbon (C) BENZENE (similar to the other 2 aromatic amino acids (AAAs) phenylalanine (Phe, F) & tyrosine (Tyr, Y). But Trp has a second “unique” ring we – a 5-membered PYRROLE. Pyrole is classified as HETEROCYCLIC because it has an atom “different” than “the us” carbon. I’m not sure how to abbreviate “yoush” in text, but what I’m trying to tell you is that a nitrogen (N) snuck in. BUT it’s STILL AROMATIC! And bulky and uncharged, so it’s in the category of LNAAs (Large Neutral Amino Acids), a class which includes Tyr, Phe, Ile, Leu, Val, and Met. We’ll get back to this later because all of these guys have to fight for the same doors to get into cells (LNAA transporters).
That N that snuck in is able to join the commune because it has a loan pair of e⁻ take “role” of double bonds in resonance structures (see yesterday) in providing “extra” e⁻. So, all in all, you get a total of 10 “extra” e⁻ shared throughout 2 rings. And this is ultra-stabilizing – it’s like an electron play-date where the atoms can get help reigning in their energetic electrons.
And the aromatic-ness makes it “visible” in the “invisible” range. As a protein biochemist, I work on figuring out thing like how tiny parts of tiny proteins affect how they interact with other tiny things like other proteins or nucleic acids (DNA or RNA). They’re all way too small to see, so I need an alternative way to “look” at them.
Spectroscopy uses light to measure things by taking advantage of different molecules’ tendency to absorb certain wavelengths of light to different extents (e.g proteins absorb strongly at one wavelength and DNA absorbs strongly at another). So, basically, if you shine light through a solution and then look to see what light makes it through you can infer things about what was in that solution. The more light that’s “missing,” the more that was absorbed and thus the more of that molecule there was.
This comes in really handy. For example, we have a UV detector that monitors what comes off protein-purification columns so you can tell where your protein comes off. And we often use a NanoDrop spectrophotometer to measure concentration of molecules like nucleic acids.
In addition to concentration, a lot of information about purity can be gained by looking at where they *shouldn’t* be absorbing much light. The height of the peak you usually focus on for a particular molecule is where it absorbs best -> this corresponds to how much stuff is there (concentration) whereas ratios between peaks can tell you about how pure that stuff is.
But how does all this actually work? And how does Trp fit in?
Light (electromagnetic radiation) (EMR) is little packets of energy traveling in waves. Different colors have different wavelengths of light with different energies (this is also true for “invisible” colors – wavelengths outside of the visible spectrum – like radio waves, which are lower frequency and ultraviolet (UV) waves which are higher frequency. Different molecules absorb different wavelengths of light to different extents, and this can be quantified by a number called the extinction coefficient (ε), which tells you how well a molecule absorbs light of a particular wavelength. More on *why* here: http://bit.ly/2CfaXbJ You can then use this equation called Beer’s law to figure out how much of a protein or DNA or anything that absorbs based on it
But TLDR, it has to do with how much energy the outer electrons of the molecule have and how much more energy they need to get to the next level – electrons can be thought of as living in “houses” called molecular orbitals – it’s not that they “always” live in one place – they’re constantly zipping around, but these orbitals are where you have the greatest chance of finding them. Orbitals farther from the nuclei of the molecules (where the positive protons and neutral neutrons are held) require electrons to have higher energy to live there. If a molecule gets hit by a photon of the optimal energy, the photon can get absorbed and its energy used to promote a lower energy electron to a higher energy, further from the nucleus, orbital.
Electron delocalization through resonance involves the merging of some neighboring molecules’s orbits (in the case of armomatic rings, they merge into that shared donut). And this lowers the cost to move to promote an electron.
All proteins absorb *some* UV. Proteins peak at 280 & 230. The 230nm absorbance is from the generic backbone part – corresponds to absorbance by the peptide bonds linking the letters (those have some resonance too remember). The parts of proteins that absorb at 280 are aromatic rings (sound familiar?) Only 3 of the 20 common amino acids have these, and different proteins have different numbers of these 3 amino acids. So different proteins absorb 280nm light differently, which is reflected by different extinction coefficients.
Since Trp is the main absorber at 280, the the UV280 absorbance per protein is gonna depend mainly on how many Trps that protein has. “Abnormal” Trp numbers can trip you up! “Too many” and you can underestimate and “too few” and you can overestimate protein concentration if you go by the “average,” but if you know the protein’s sequence you can calculate the “estimated extinction coefficient” for your exact protein, using free online software tools like Expasy ProtParam, which you can stick into Beer’s law. I say estimated because context matters – the local environment around the absorbing part can influence how eager it is to absorb a photon. much more here: http://bit.ly/bradforduv
DNA absorbs really strongly at UV260, where protein doesn’t – and it absorbs so strongly that it’s relatively easy to see if you have DNA in your protein prep, but it’s harder to tell if you have protein in your DNA prep – 260 will dominate the 260/280 ratio
In addition to helping us see proteins in the chromatograph, Trp helps us orient our models in x-ray crystallography. This is a technique where we look at protein structures by bouncing x-rays off molecules’ of molecules’ e⁻ , then capturing those bounced-off x-rays on a detector and working backwards from the pattern of spots (diffraction pattern) to find the bounce-off points. Trp has lots of concentrated e⁻ so it diffracts really well & it’s really big so you get a powerful signal you can use to help orient your protein model. http://bit.ly/xraycrystallography2
Yesterday we looked at some chemical signalers made from the amino acid tyrosine. Tyr gives us dopamine, adrenaline & noradrenaline (catecholamines), & thyroid hormones. Trp also gives us signaling products – melatonin (a hormone) & serotonin (a neurotransmitter), and this is where we get into the turkey myth.
But first let’s recap on what those messenger “job descriptions” mean. Your body has billions of cells and they need ways to talk to one another. Since they provide direct connections, nerves are great if you want to send a signal to one specific place. Nerve cells called neurons communicate with other neurons and/or muscle, gland, etc. cells by passing messages really short distances. One neuron releases a chemical called a neurotransmitter (there are lots of different ones, including noradrenaline) into the gap between the 2 of them (synapse) and then receptors on the second one bind that neurotransmitter and relay a message into the cell.
But what if you want to send a message to your entire body? Unlike the nervous system, which requires all the “wiring” to be laid out, like having one of those “direct” phone lines, which are great if you only want one person to get your message (and no wiretaps allowed!), the ENDOCRINE SYSTEM is more like radio. It sends a message out to everyone, but you can only get it if you have your radio tuned in to the right wavelength. Instead of sending out waves, the endocrine system sends out chemical messenger molecules called hormones from “broadcast stations” called glands and only cells expressing the matching receptors can hear the message and respond (with various responses depending on the type of cell getting the broadcast).
Hormones get secreted into the bloodstream so they pass by all your cells but only cells with the right receptors can respond (like having your radio tuned to the right wavelength). These receptors are proteins that are embedded in your cells’ membrane. Like all proteins, the instructions for making them are written in your DNA “blueprint” – so each cell has the instructions and thus could make it if they wanted to but, thanks to lots of regulation, only certain cells do. And they can make more or less at different times to “ramp up” or “calm down” sensitivity & responses to signals.
So, that’s the gist of hormones and neurotransmitters in general, but what about Trp in particular? Trp can be converted into serotonin (5-hytdroxytryptamine (5-HT) which can act as a neurotransmitter that lets brain talk to other cells to regulate things like mood, appetite, sleep regulation, bone metabolism, and GI motility. And, in the pineal glad, it can be further converted into melatonin (N-acetyl-5-methoxytryptamine). As the “gland” in pineal gland suggests, melatonin is a hormone. It helps regulate sleep & wakefulness
It’s only made in the pineal gland and the retina but not other places in your body because they don’t make the needed enzyme. The pineal gland is a tiny gland in the middle of your brain and it secrete melatonins into blood & cerebrospinal fluid (brain juice). This synthesis & secretion increases during the dark period of day, so you get sleepy at night.
And you get the turkey makes you sleepy myth you probably heard around Thanksgiving. So let’s tackle it. 1st of all, turkey doesn’t have any more tryptophan than other meats (cheese actually has even more). 2nd of all, when you eat Turkey at Thanksgiving, you’re probably eating a lot more than just Turkey – likely you’re also eating a lot of carbs. And this leads to the release of a hormone called insulin. It’s mainly known for its role in telling cells to let in and use glucose (blood sugar) (it’s either not produced enough or not well-recognized in patients with diabetes). But it also has the role of telling cells to let in LNAAs (Large Neutral Amino Acids).
These include a lot of the ones we’ve seen recently – Tyr, Phe, Ile, Leu, Val, and one we’ll get to in a later day, Met. It also includes Trp, but in your blood that Trp’s pretty tied up. Turns out that indole ring is also good at binding to serum albumin, an abundant protein in your blood. So Trp has a hard time getting in, but the others are free-er so they can get into your muscle cells, etc. Leaving Trp with less competition when it gets to the brain and has to cross the blood-brain barrier. It has to compete with all those other guys here too, but you’re starting with an advantage and it likes the transporter here better. So more Trp gets taken in and used. And, although this situation might lead to increased Trp crossing the blood-brain barrier into the parts of your brain where serotonin is made, those parts don’t make the enzymes you need to go all the way to melatonin.
And the part that does have those enzymes, the pineal gland, is actually not protected by the blood-brain barrier. So it’s seeing the same “more competition to get in” blood as the rest of your body. The biggest part of the tiredness is probably just your metabolism working really hard to try to breakdown your big meal. And potentially some alcohol consumption…
Another time you can see changes in serotonin production is when you’re stressed. In this case, *less* Trp gets turned into serotonin because stress hormones cause Trp to be channeled into different pathways like the kynurenine pathway which breaks down Trp a bit before it builds new things with its parts. The first step is breaking open that indole ring – done with the help of the enzyme tryptophan 2,3-dioxygenase. This gives you N-formyl-kynurenine which is converted (by arylformamidase (aka kynurenine formamidase) to kynurenine, which is a branch point. From there, Trp has different fates including being used to make the electron transport molecule NAD+ and the amino acid alanine, as well as some other brain-signaling molecules.
I’m not going to go into all of them, because Trp has the most complex catabolism (breakdown) pathways of any amino acid, which I guess isn’t surprising since it’s got the most stuff to start with. Some of its breakdown products can be used to make sugars (so we call it glycogenic) and some can be used to make ketone bodies and fats, so it’s also characterized at ketogenic. We can make a lot of things from it, but we can’t make *it* – so we have to eat it “pre-made” in our food (we call such amino acids essential). If you’re a vegetarian like me, don’t worry – non-meat protein sources, like the milk protein casein it was 1st isolated from by Frederick Hopkins in 1901, nuts, & chocolate.
Another way we can characterize Trp is NONPOLAR – the hydrocarbon nature, with its equal charge spreading, dominates, but the N also makes it less hydrophobic than phenylalanine. Because the N doesn’t just have that electron pair it’s sharing – it also has an H, and it doesn’t share fair with that H, so the H becomes partly positive and that can get attracted to the partly negative Os in water, so it can “hydrogen bond” with the water and thus it won’t freak out if it’s exposed on the surface.
It was named because it was found in proteins cut by “trypsin” – it was actually named before they figured out what it was – Neumeister gave the name to the thing they found when they cut proteins that made colored reactions happen in 1890. https://doi.org/10.1021/cr60033a001
how does it measure up?
coded for by: UGG it only gets one!
chemical formula: C11H12N2O2
molar mass: 204.229 g·mol−1 “W”owsa!