Well deserved, Roger Tsein, Osamu Shimomura and Martin Chalfie will share the prize for their work on GFP.  The work is seminal in many regards, not the least of which is because it makes shit glow and glowing is way cooler then Pd couplings.  It’s important to note that I think Tsein should STILL RESPOND TO EMAILS no matter how many stupid fucking prizes he wins.  But that’s fine.

GFP is a very interesting molecule with amazingly bizarre chemistry at its core.  Central to the protein is a fluorphore – a conjugated heteroaromatic cluster – that emits green light.  Roger’s first publication on the subject was in 1995, iirc, so it took him a little more than 10 years to snag the highest honor in sciences for his efforts.  The detailed mechanisms of the actual biosynthesis have been remarkably well done and so, it is not unusual that such a discovery, which took less than 10 years to go from the lab to glowing mammals, should take a little more than that to go from obscurity to a Nobel Prize.

Above is an interactive X-Ray structure of GFP (that image was captured from my computer. You CAN find the chromophore, but you have to look for it.) In any regard, I find a good crystal structure to be refreshing and enjoyable. So click on that image up there and load the structure! You’ll notice a ton of fucking water. I wish I were more of an expert at JMOL so I could just have the protein up there, but it’s a sufficiently intuitive program that you should be able to explore the structure without great confusion.

It has been a while, but I knew that the AMAZING quality coming out of the journal of Food Chemistry would get me to come back for more.  Indeed, a great one from Yaguang Luo (DOI: 10.1016/j.foodchem.2008.02.070) taught me just oodles of facts:

Enzymatic browning is a widespread color reaction occurring in fruits and vegetables and tea leaves. The browning reaction requires the presence of oxygen, phenolic compounds and polyphenol oxidases (PPO) and is usually initiated by the enzymatic oxidation of monophenols into o-diphenols and o-diphenols into quinones, which undergo further non-enzymatic polymerization leading to the formation of pigments. Although enzymatic browning
is beneficial to the color and flavor development of certain food items such as tea, coffee and cocoa, it impairs the quality and salability of fresh-cut produce. A variety of fruits and vegetables, such as lettuce, potato, apple, pear, banana and peach, are susceptible to enzymatic browning during processing and storage.

That’s interesting and about where the paper’s utility turns more toward being the recipient of my brown post consumption compost from my pooper then scientific document.  One thing is for sure though, the world needs less brown fruit. Indeed, 20% of West Virginian Democrats would not eat brown fruit based simply on color. Therefore, preventing browning is a must, and can we do it? Yes we can. But Yaguang Luo has a little more than just Hope on his (or her) side… standing with him (or her) was a compound called sodium chlorite. While most “anti-browning” agents are reducing agents – think ascorbic acid this one is an oxidant a bleaching agent if you will. But how is this bleach-like substance keeping vegetables from turning brown? QUICK!  To the Science Machine! Allow me to elaborate their experimental procedure:

Exp 1. Professor Luo incubated protein and found that at oxidant concentrations greater than 1.5 mM, protein no longer worked. PROOF OF INHIBITION OF PROTEIN! NOBEL PRIZE IN DA MAIL! Next, will concentrated sulfuric acid inhibit protein? Maybe another FOOD CHEMISTRY article if so.

Exp 2. Professor Luo mixed oxidant with browning shit and no browning occurred. Surprising result, since I didn’t know BLEACHES remove COLORED COMPOUNDS by OXIDATION. OMFG. NOBEL PRIZE NUMBUH TWO!

So, there you have it. An oxidant removes colors and denatures inhibits proteins. But still, learning about how food turns brown made it totally worth the read.

This isn’t C&E news concentrates, but I want to cram a few in that I missed over the last few months. I’ve read and loved and you’ll love these advances in the field of chemistry:

First: DOI: 10.1126/science.1152692. Fucking brilliant piece from the Baker lab representing state of the art fuckability of proteins. The title: “De Novo Computational Design of Retro-Aldol Enzymes” gives you a half assed idea of what’s actually going on. Where the typical “state of the art” to select for proteins that do specific chemistry is to essentially make a fuck-ton of proteins and run them on a specially designed column or do some Systematic evolution of ligands by exponential enrichment. The idea is NOT to go and design an active site of a protein and then cut out the active site of another protein and stick your active site in it to do the chemistry you want it to do. My brief description trivializes the difficulty inherent here and may overstate the utility, but it’s a monumental push forward for protein engeneering that relies not upon random insertions or specific point mutations but de novo design of an active site and then finding a protein that is compatible with it.

Second: DOI: 10.1088/1468-6996/9/1/014104. Sir Fraser Stoddart and Bill Goddard collaborated to create one of the most awesome ideas in supramolecular chemistry , which I’ve gushed over here. The paper details the attempt and successful creation of the “quasi-tristable [2]catenane.” Limitations exist in the system and it’s apparent that the design, as it stands, is unlikely to produce the desired results as an unfortunate overlap appears to exist between the absorption and CT bands of the various colored bits, but I’m still undaunted in my interest of a system like this.

Finally: DOI: 10.1002/anie.200800891. The prior post makes this next post a bit more apropos. Design of rotaxanes with strong binding interactions necessarily makes “shuttling” them around rather difficult but the synthesis of interlocked molecules nearly necessitates strong interaction between thread and macrocycle to assemble. A Catch 22, as it were. Leigh and Zerbetto have a rather clever hack which is slightly reminiscent of some of Vogtle’s (and Leigh’s own) work, where the macrocycle is used to direct the chemistry that is going to make the rotaxane in the first place, though they use it in a distinctly clever way by exploiting the copper mediated Cadiot–Chodkiewicz reaction. I have pretty high hopes for this sort of thinking in the design of molecular shuttles.

These are just a few of the articles I read and found very interesting. If I weren’t so goddamn busy I’d give literature more treatment and limit my fluff posts. But, it’s my blog, so blah.

Unremarkably, I was totally stuck at O’Hare last night until midnight waiting for a flight that was supposed to take off around 6:00. This being the second time in less than 30 days that O’Hare made me want to die. I mean… Fuck O’Hare. Anyway, given my late arrival and sleeping in later than normal, I’m sitting around with little to nothing to do waiting for mummy Finchsigmate to arrive home with delicious scrambled caviar and figured I’d do a little post on a drug most people probably haven’t heard of.

Cerezyme is freeze dried imiglucerase, a recombinant (and modified) form of the enzyme Glucocerebrosidase and it costs about $200,000 a year for life. It’s one of three treatments for Gaucher’s disease which doesn’t involve scooping organs out with a spoon before they explode or topping the ‘ol femurs off with some new bone marrow. (The others are Miglustat, a small drug and Ceredase, an enzyme derived from leftover placenta… fuckin’ gross.) In general, you don’t have to worry if you aren’t an Ashkinazi Jew or a Sweed from Norrbotten, as they are the ones that seem to be afflicted.

The drug is made inside CHO cells, which are just Chinese hamster ovary cells that have been transfected to produce imiglucerase. Imiglucerase is not the real deal; since they do not produce the actual Glucocerebrosidase, 15% of patients develop antibodies to the drug and of those about half have an allergic reaction. My understanding is that the modifications, specifically the Mannose sugars on the terminus of the existing polysaccharide chain, lead to a selective uptake of the enzyme by macrophages that are resent in liver, spleen and skeleton. Without this modification the drug does not work effectively.

The drug works just like the natural protein by catalyzing the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide as part of the normal degradation pathway for membrane lipids.

If you want to make your own, here is the sequence:

ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANH
TGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIR
VPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWT
SPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGL
LSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPE
AAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRG
MQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHL
GHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFL
ETISPGYSIHTYLWRRQ + manoses

Why is it $200,000 a year? Maybe more, depending on your dosage needs? Certainly not all recombinant drugs are this expensive. Insulin has been grown in bugs for almost a decade and it’s dirt cheap in comparison.

The answer is that, quite simply, there are probably only 10,000 people in the world with this disease and 5,000 of them are already on it. To recoup that investment they needed to put the price point higher. But it’s an orphan drug! They already got a shitton of money back from the gubment for just making it, why do they need to sell it for such a shitton of money it actually has sales in excess of a billion dollars a year?

Well, that’s one of them sticky ethical questions that the ChemBlog is running out of time to answer… Caviar is almost ready.

A new paper produced by the Mayer group has the biological-molecular-probe community abuzz (JACs ASAP FREE AUTHOR CHOICE!). Everyone knows soon-to-be-Nobel-prize-winner Roger Tsien and his magical collection of multicolored fluorescent proteins. They are, of course, gigantic proteins (though they assemble themselves, which is the future, IMHO). Indeed, many of the current methods to create in vivo labels rely upon the assembly of large proteins which are conjugated to the protein of interest.

I haven’t rocked out the shitty quality of MS Paint in a while, and this is the perfect opportunity. See, it works like this. Option 1 is to just allow the cell to make the whole dye. You transfect the cell to append the correct genetic sequence to make GFP, and the cell makes it for you. No fuss, no muss. Option two is more two steppy. You can use brighter dyes, however. A “HaloTag” protein (a hydrolase mutant) is expressed just like GFP, but it binds selectively to a synthetic “Halotag ligand.” That’s neat, I guess… if you suck. The final option, which requires no gigantor protein expression, is one using biarsenical fluorescent dyes which selectively bind to cystines exposed on alpha helices (again transfected). This last method still may require exogenous shit to dangle off your protein, but it’s small. Of course, cystines are nucleophilic, easily oxidized and generally reactive amino acids, but you can’t expect miracles.

Mayer’s work builds upon previous work which used weaker fluorescein dyes with cutesy little names like FlAsH. The cyanine dye series (specifically the very stable Cy3) was used by Mayer as an alternative and/or compliment to the cutesy named dyes, all of which bind to the same genetic sequence (CCXXCC), which is roughly one helical turn. They Cy3 dye is longer, which means the arsenics are further apart, so it would bind to a different sequence (CCXXXXXCC). Thus, the two dyes are orthogonal binders and can be used for FRET experiments!

In short, Mayer has made the biarsenical multiuse probes a useful tool in protein-protein interactions via FRET and kicks ass for paying the brazillion dollars to make this article free to the public as an author’s choice.