Tag Archives: green fluorescent protein

Everything not expressly forbidden biochemically is happening somewhere

A fairly oblique introduction (from an earlier post)

Sherlock Holmes and the Green Fluorescent Protein

Gregory (Scotland Yard): “Is there any other point to which you would wish to draw my attention?”
Holmes: “To the curious incident of the dog in the night-time.”
Gregory: “The dog did nothing in the night-time.”
Holmes: “That was the curious incident.”

The chromophore of green fluorescent protein (GFP) is para-hydroxybenzylidene imidazolinone. It is formed by cyclization of a serine (#65) tyrosine (#66) glycine (#67) sequential tripeptide. It is found in the center of a beta barrel formed by the 238 amino acids of GFP.

What is so curious about this?

Simply put, why don’t things like this happen all the time? Perhaps nothing quite this fancy, but on a more plebeian level consider this: of the twenty amino acids, 2 are carboxylic acids, 2 are amides, 1 is an amine, 3 are alcohols and one is a thiol. One might expect esters, amides, thioesters and sulfides to be formed deep inside proteins. Why deep inside? On the surface of the protein, there is water at 55 molar around to hydrolyze them purely by the law of mass action (releasing about 10 kJ/Avogadro’s number per bond in the process). Some water is present in the X-ray crystallographic structure of proteins, but nothing this concentrated.

The presence of 55 M water bathing the protein surface leads to an even more curious incident, namely why proteins exist at all given that amide hydrolysis is exothermic (as well as entropically favorable). Perhaps this is why proteins contain so many alpha helices and beta sheets — as well as functioning as structural elements they may also serve to hide the amides from water by hydrogen bonding them to each other. Along this line, could this be why the hydrophilic side chains of proteins (arginine, lysine, the acids and the amides) are rather bulky? Perhaps they also function to sterically shield the adjacent amides. After all, why should lysine have 4 CH2 groups to separate the primary amino from the alpha carbon? Ditto for the 3 CH2 groups separating the guanidine group, and the 2 CH2 for glutamic acid.

We now have an example before us of an ester between threonine and glutamic acid within the same protein. For details see Proc. Natl. Acad. Sci. vol. 111 pp. 1229 – 1230, 1367 – 1372 ’14. It is put to use to stabilize long thin proteins subject to mechanical stress. All sorts have bacteria have little hairs (pili) allowing them to attach to our cells. The first example were found in some nasty characters (Streptococcus progenies, Clostridium perfringens), possibly because they’re under intense study because the infections they cause are even nastier. Interestingly, the ester is buried deep in the protein where water can’t get at it so easily. This type of link on external proteins turns out to be fairly common in Gram positive organisms.

So everything not biochemically forbidden is probably happening somewhere.

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