Aromatic rings are planar in proteins aren’t they? More trouble for computational chemistry

Every organic chemistry book worth its salt has a diagram of the heats of hydrogenation of Benzene (the new Clayden has it on p. 158).  Adding H2 across a double bond releases energy, because saturated hydrocarbons have a lower energy than alkenes.  However the heat of hydrogenation of benzene is some 208 kiloJoules/mole, which is considerably less than 3 times the heat of hydrogenation of cyclohexene (3 * 120 kiloJoules/mole).  Then we’re off for a romp through the planarity of benzene allowing the p orbitals to overlap, the Huckel rules etc. etc.  It’s why benzene (and the aromatic nucleotides making up DNA and RNA) are flat — move one of the atoms out of the plane, and you decrease overlap, raise energy, etc. etc.

Except that there are 19 proteins where this isn’t the case for the 6 membered rings of phenylalanine or tyrosine.  A truly fascinating paper [ Proc. Natl. Acad. Sci. vol. 109 pp. 9414 – 9419  ’12 ] describes alpha-lytic protease (alphaLP from here on) in which phenylalanine #228 has a bent benzene ring.  Even more interestingly, this raises the energy of the protein and appears to be an integral part of the protein’s biological functioning.   It’s not an accident.

When you do Xray crystallography, 2 Angstrom resolution is usually enough to show you what’s going on (the C – C bond is 1.54 Angstroms).  However, ultrahigh resolution structures (resolutions under 1 Angstrom) have become available for some 100 proteins, allowing you to see if aromatic rings are truly flat.

Phenylalanine #228 of alphaLP is not flat at all, deviating by 6 degrees from planarity.  How much is this?  Well the benzene carbon carbon bond length is 1.4 Angstroms, so it’s 1.4 + 2 * 1.4  * sine (30) = 1.4 + 2 * 1.4  * 1/2 = 2.8 Angstroms from carbon 1 to carbon 4.  How far does 6 degrees takes carbon 4 out of the plane of carbons 1, 2 and 6? It’s 2.8 * sine 6 degrees.  Since sine 6 degrees is .10, this means that carbon 6 is only .28 Angstroms out of the plane — high resolution indeed.

Now it gets interesting, from both a chemical and biological point of view.  It turns out that, purely on an energetic basis, the unfolded form of alphaLP is 4 kiloCalories/mole lower in energy than the folded (native) form, so the native form is metastable.  However, it is kinetically stable, with a half-life for unfolding of 1.2 years, a classic example of a kinetically stable, thermodynamically unstable chemical entity.

It gets more interesting (and confusing to me) because the folding barrier is said to have a half-life of 1,800 years (4 kiloCalories/mole shouldn’t make that much difference should it?).  Does anyone out there know why the folding and unfolding barriers should be so different.  So how does the protein get into the native configuration?  By a covalently attached folding catalyst (called the pro region), which is removed when the native state is reached.  Kinetic stability seems to exact a toll on the difficulty of folding, one which selection is willing to pay.

Now it’s time to look at the environment of phenylalanine #228.  The ring is being pushed out of shape by threonine #181 below and tryptophan #199 above.  So the authors did the obvious, replacing Thr181 by glycine and then alanine and watching what happened.  The mutants unfolded faster — so the distortion in some way is raising the energy of the transition state, and thus is functionally important in the kinetic stability of the protein.   The authors are silent as to the actual structure of the transition state for unfolding, but rates are rates and their conclusions seem sound.  As they say in computer land, that’s not a bug, that’s a feature.  Why would you wan’t alphaLP to be kinetically rather than thermodynamically stable?  The authors think that kinetic stability makes alphaLP  better able to survive in harsh environments.  Perhaps

Well, how common is this?  There are some 100 protein structures now available at ultrahigh resolution.  19 of them have nonplanar aromatic side chains by 6 degrees or more (see figure 5 p. 9418).  Who’d a thunk it.  One wonders how many structures were thrown out because everyone knew that aromatic rings are planar.

What does this mean for the computational chemist?  The low energy form may not actually be the important one.  What we’ve assumed about side chains may not be true.  It makes the protein folding problem even more complicated.

They don’t discuss tryptophan planarity.  Clearly more ultrahigh resolution studies of proteins are needed.  Think of the decades spent studying proteins, and here’s something brand new.  Reading the scientific literature is like reading a Russian novel with thousands of new characters popping up and doing  the unexpected.

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  • MJ  On June 25, 2012 at 12:45 am

    Disclaimer – This is one of those topics I never tire of beating to death.

    So, I haven’t examined the structures, nor have I read the paper. I did quickly check the PDB files before starting in on this comment. The structures they list in their paper were all obtained at 100 K in a beamline. I have commented more than once about cautions one must take with examining protein structures determined under cryogenic conditions. My general reaction to finding out that there might be strange, counterintuitive features observed in structures derived from cryocrystallography is a mixture of amusement and boredom, as it’s hardly new to me at this point in time.

    I should probably read the paper, though.

  • luysii  On June 25, 2012 at 7:33 am

    Please do, and let me know what you think. It seems to be more than a quirk, as there is some biologic relevance to the distortion, as evidence by the mutation results on unfolding. I’ll be away for a week or so starting tomorrow.

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