What's an Iodide doing stabilizing a helix?

One of the most important- and least understood- effects dealing with biomolecular structure concerns the effects of salts on protein conformation. The famous Hofmeister Series for ions that either 'salt-in' or 'salt-out' proteins is well known, but the mechanism through which the ions act is controversial and probably involves not one mechanism but different ones under different circumstances.

In an interesting single-author JACS paper, Joachim Dzubiella studied the effects of different salts of sodium and potassium on the structure of alpha helices in solution. Even something as common and widely studied as the alpha helix is still an enigma. For example, the simple question "What contributes to the stability of an alpha helix?" is controversial and not fully answered yet. In this context I will refer the reader to an excellent perspective written by Robert Baldwin at Stanford that tries to answer the rather simple question: "How much energetic stability does a peptide bond in a helix contribute?". Baldwin looks at two approaches to understand the problem. One is the 'hydrogen bond inventory' approach which simply lists the bonds broken and formed on each side when an amide group desolvates and forms a peptide bond. Based on this approach, the mean figure for peptide h-bond energy has been estimated as 0.9 kcal/mol/h-bond. Even though this quantity is small, a 100 residue protein where 70 residues form hydrogen bonds is clearly going to lead to a very substantial net stabilization. The second approach that Baldwin considers is the electrostatic solvation enthalpy or free-energy method, where one uses the Born equation to estimate the strength of a h-bond. Using this approach Baldwin gets a very different answer- 2.5 kcal/mol. Clearly there is still some way to go toward estimating how much peptide h-bonds contribute to stability. One important factor not considered by Baldwin is the entropy of the water. Another important factor that he does consider is the preferential desolvation for helix formation that depends on the exact residues involved. We have ourselves encountered desolvation issues in continuing work on amyloid beta-sheets.

But back to Dzubiella's paper. Dzubiella uses MD simulations to study the dynamics of helix-salt interaction. He considers helices where a i---> (i+4) salt bridge between the side chains of a glutamate and lysine has stabilized the conformation. He looks at which salts stabilize helices and which ones destabilize them. From these detailed simulations he gains some valuable insight into the radically different behavior of rather similar ions. For example, K+ ions are much less able to destabilize helices than Na+ ions. This is due to preferential interaction of carboxylate groups involved in salt-bridge formation by Na+. Due to its smaller size, Na+ is better able to interact with carboxylates than K+.

However, we have to remember that Na+ or K+ or any of the other ions have to compete with water when interacting with amino acids in the peptide. Water is in great excess and water also efficiently interacts with carboxylates (1). The MD simulations reveal that a curious and unexpected helper comes to the aid of the Na+ ions- I- ions. Iodide interestingly interacts with the non-polar parts of the peptide, thus "clearing" water away and paving the way for Na+ to access the carboxylates and carbonyls. This unexpected observation again sheds light on the different properties of iodine compared to the rest of the halogens (2). Iodide is much bigger, has a diffuse charge and is therefore much more polarizable. Apparently it is so electronically watered down that even carbon thinks it is harmless and can preferentially interact with it.

This curious observation tells us that we know less about the elements than we think. From the observation of weak hydrogen bonds and halogen bonds to the unexpected non-polarity of iodide, surprises await us in the realm of biomolecular structure and indeed in all of chemistry. It is also thanks to tools like MD that we can now gain insights into the details of such molecular interaction.

Notes:
(1) In fact water can interact so well that it might steal a few h-bonds from the peptide and destabilize the helix. That's why trifluoroethanol (TFE) or hexafluoroacetone are so good at stabilizing helices (these lead to "Teflon-coated peptides"), because the fluorine cannot steal h-bonds from the peptide backbone.

(2) For example iodide most efficiently forms halogen bonds with oxygen, a phenomenon now well-accepted.

References:
Joachim Dzubiella (2008). Salt-Specific Stability and Denaturation of a Short Salt-Bridge-Forming α-Helix Journal of the American Chemical Society DOI: 10.1021/ja805562g

R. L. Baldwin (2003). In Search of the Energetic Role of Peptide Hydrogen Bonds Journal of Biological Chemistry, 278 (20), 17581-17588 DOI: 10.1074/jbc.X200009200

MD? Check...at least in this case

I have often talked on this blog about the fallacy of deriving single average conformations for molecules from average NMR data. How can a molecule with ten rotatable bonds (and frequently more) have any single or dominant conformation in solution? And yet, one repeatedly comes across publications dealing with single average conformations derived for organic molecules.

Peptides, being favourite targets for solution conformational analysis, are often subjected to such analysis. One comes across many instances where people want an alpha helix, or a 3/10 helix, or a gamma turn. They then design a peptide which they think is constrained to form that motif. They get their average NMR data (coupling constants and distances from NOE data), and then do a constrained molecular dynamics run, and see their favourite motif on the screen. But of course, what you put in is what you get out. If you are constraining the molecule to fit the NMR data in the first place, it's not going to stray away from it, and not surprisingly you see what you want to see (Isn't this a general theme and trap for so many of us...to be tempted to see what we want to see?).

In general, molecular dynamics is not a very efficient method of searching conformational space. Plus, every MD program uses some force field, and force fields are well-known to give fairly accurate geometries if well parametrized, but not good energies. So there is no guarantee that all the "low-energy" conformations of any molecule will be sampled. But in theory, if one could actually search all of conformational space with a good force field, then one would explore the whole ensemble of structure that a molecule adopts in solution. Even with faster computers though, that goal is going to unrealizable for a general situation.

However, MD can be a good way of exploring conformational space if the molecule is highly constrained in the first place. In such a case, it can serve as a good way of deconvoluting the average NMR data. And this is prcisely what Nikiforovich and Marshall, two researchers who do seem aware of the general problem of assigning multiple conformations, have done (J. Med. Chem, DOI: 10.1021/jm070084n). They have taken a cyclic pentapeptide, Pro-Ala-Ala-Ala-Ala, and done long 100 nanosecond (which is a huge amount of time for a MD run) on the molecule. Most importantly, their MD runs are unconstrained. After running the MD with two different force fields and different starting velocities, they consistently see the same results; a major conformer without a gamma turn, and a minor one (about 6%) with a gamma turn, a usually forbidden motif. They also gratifyingly note that the average distances from their ensemble of MD structures match well with the NMR data.

This analysis was feasible and realistic because of a couple of reasons; firstly, the molecule is small (although it still has 8-10 rotatable bonds) and one can actually do a 100 ns MD run on this. Secondly, they used many different conditions that converged to the same two conformational profiles, and thirdly they used unconstrained MD to remove any bias. I am not completely satisfied with it though, because the molecule still does have 10 rotatable bonds and is quite flexible. But it is a ring, and some of the bonds are not as flexible, so in general it is constrained and I think the conclusions are quite believable.

For once, it gave me a satisfied feeling to see an analysis that gave results that are quite likely to be true, complete, and sweet.

P.S. Garland Marshall also has one of the more memorable quotes in science that I have heard:

"Nature does not shave with Ockham's Razor!"