Overturning hydrophobic assumptions

One of the most fun things about chemistry is that for every laundry list of examples, there is always a counterexample. The counterexample does not really violate any general principles, but it enriches our understanding of the principle by demonstrating its richness and complexity. And it keeps chemists busy.

One such key principle is the hydrophobic effect, an effect with an astounding range of applicability, from the origin of life to cake baking to drug design. Textbook definitions will tell you that the signature of the "classical" hydrophobic effect is a negative heat capacity change resulting from the union of two unfavorably solvated molecular entities. The nonpolar surface area of the solute is usually proportional to the change in heat capacity. The textbooks will also tell you that the hydrophobic effect is favorable principally because of entropy; the displacement of "unhappy" water molecules that are otherwise uncomfortably bound up in solvating a solute contributes to a net favorable change in free energy. Remember, free energy is composed of both enthalpy and entropy (∆G = ∆H - T∆S) and it's the latter term that's thought to lead to hydrophobic heaven.

But not always. Here's a nice example of a protein-ligand interaction where the improvements in free energy across a series of similar molecules comes not from entropy but from improved enthalpy with the entropy actually being unfavorable. A group from the University of Texas tested the binding of a series of tripeptides against the Grb2 protein SH2 domain. The exact details of the protein are not important; what's important is that the molecules only differed in the size of the cycloalkane ring in the central residue of the peptide- going from a cyclopropane to a cyclohexane. They found that the free energy of binding improves as you go from a 3-membered to a 5-membered ring but not for the reason you expect, namely a greater hydrophobic effect and entropic gain from the larger and more lipophilic rings.

Instead, when they experimentally break down the free energy into enthalpy and entropy using isothermal titration calorimetry (ITC), they find that all the gain in free energy is from enthalpy. They find that every extra methylene group contributes about 0.7 kcal/mol to the interaction. In fact the entropy becomes unfavorable, not favorable as you move up the series. There's another surprise waiting in the crystal structures of the complexes. There are a couple of ordered water molecules stuck in some of the complexes. Ordered water molecules are fixed in one place and are "unhappy", so you would expect these complexes to display unfavorable free energy. Again, you would be surprised. It's the ones without ordered water molecules that have worse free energy. The nail in the coffin of conventional hydrophobic thinking is driven by the observation that the free energy does not even correlate with decreased heat capacity, something that's supposed to be a hallmark of the "classical" hydrophobic effect.

Now it's probably not too surprising to find the enthalpy being favorable; after all as they note, you are making more Van der Waals contacts with the protein with larger rings and greater nonpolar surface area. But in most general cases this value is small, and the dominant contribution to the free energy is supposed to come from the "classical" hydrophobic effect with attendant displacement of waters. Not in this case where enthalpy dominates and entropy worsens. They don't really speculate much on why this may be happening. One factor that comes to my mind is the flexibility of the protein. The improved contacts between the larger rings and the protein may well be enforcing rigidity in the protein, leading to a sort of "ligand enthalpy - protein entropy" compensation. Unfortunately a comparison between bound and unbound protein is precluded by the fact that the free protein forms not a monomer but a domain-swapped dimer. In this case I think that molecular dynamics simulations might be able to shed some light on the flexibility of the free protein compared to the bound structures; it might especially be worthwhile to do this exercise in the absence of the apo structure

Nonetheless, this study provides a nice counterexample to the conventional thermodynamic signature of the hydrophobic effect. The textbooks probably don't need to be rewritten anytime soon, but chemists will continue to be frustrated, busy and amused as they keep trying to tame these unruly creatures, the annoying wrinkles in the data, into an organized whole.

Myslinski, J., DeLorbe, J., Clements, J., & Martin, S. (2011). Protein–Ligand Interactions: Thermodynamic Effects Associated with Increasing Nonpolar Surface Area Journal of the American Chemical Society DOI: 10.1021/ja2068752

Water wires and hydrophobics

Quick survey of two interesting articles

P. Balaram's group at the Indian Institute of Science in Bangalore has turned a serendipitous observation into a nice study of a single file water wire inside a hydrophobic peptide nanotube. Based on the crystallographic data two models have been proposed for this wire. Such a structure can be the starting point for interesting MD simulations.
DOI: 10.1021/ja9038906

A group at Boston University and SLAC has found an explanation for the catalytic action of acetoacetate decarboxylase that refutes an elegant explanation provided by the famous late bioorganic chemist Frank Westheimer. Westheimer had proposed that a key lysine involved in nucleophilic attack was neutral because of proximity to another lysine; the electrostatic repulsion between two charged lysines would not favor the ionized state for both of them. The present group has obtained the crystal structure for the enzyme and finds that the two lysines are in fact far apart. Thus, electrostatic repulsion could not be responsible for the neutral nature of the lysine. Instead, using an elegant set of experiments, they find that it's being in a hydrophobic cavity that favors the lack of ionization.
doi:10.1038/nature07938

Water and Amyloid Self-Assembly

One of the most significant, interesting and yet poorly understood effect in chemistry and biology is the hydrophobic effect. An important manifestation of its role in biology is invoked by the picture of two parallel hydrophobic plates with water between them that are slowly brought close to each other. At a certain distance called the 'dewetting distance', dewetting is observed and the water suddenly gets expelled out from them. Since nature abhors the resulting vacuum, the two hydrophobic surfaces collapse and 'stick' to each other.

Bruce Berne and his colleagues at Columbia had hypothesized that such dewetting could be observed in biological systems. A remarkable dewetting transition in the protein mellitin and in other proteins in the PDB provided evidence. Now they and others investigate such possible roles of water in the formation of amyloid by molecular dynamics simulations. They focus on the central core peptide of the Alzheimer's Aß (1-42) peptide consisting of the 16-22 region. Since one of my projects involves investigation of the self-assembly of this segment, it is of particular interest to me. This oligopeptide is interesting because it seemingly is the smallest stretch of the bigger parent that also forms the characteristic cross-beta sheet structure of amyloid. Importantly, it is also soluble which means it is more amenable to structural characterization compared to the bigger peptide.

Berne's group investigates the role that water plays between two sheets of 16-22 consisting of nine peptides each. The MD simulations were run for 1 ns, a typical time for such systems. The dewetting critical distance was found to be 12.8 A. Compare this with the classic repeat distance between amyloid beta-sheets which is 9.9 A. Basically two types of phenomena were observed depending on different trajectories and conditions; dewetting, which means that water expulsion was followed by hydrophobic 'collapse', and the simultaneous occurence of expulsion and collapse. The latter phenomena can be interpreted as a kind of 'lubrication' effect that water has been hypothesized to play in the folding of proteins. They haven't been able to put a finger on which is the phenomenon in the 'real' system, but it at least seems plausible that dewetting could be observed in such systems. In the cases where dewetting is not the driving force for assembly, they dissect the interaction energies of different amino acid residues that contribute to the total energy, and find that the Phe-Phe energy contributes to the most.

From a methodological standpoint, the investigation is complicated by the fact that, as in other such studies, the results seem to depend on the methods used. This is a common characteristic of modeling and theoretical investigations. In this case, turning off the protein-water Van der Waals forces causes dewetting in every instant, perhaps not a surprising conclusion since there is now no attraction at all between the water and protein. Also, turning off the electrostatic forces caused no changes, which means that electrostatic attraction plays a small role in the system. Again probably not too surprising. A third observation is that the choice between the two phenomena depends on rather small changes in temperature.

The interpretations are also rendered ambiguous by the fact that amyloid surfaces and indeed most 'hydrophobic' protein surfaces are a poor approximation to two parallel hydrophobic plates. It should be noted that the attractive energy of interaction between two flat plates separated by a distance R is inversely proportional to the square of R. Compare this with the case of two spheres of radius R whose classical Van der Waals attractive energy is proportional to the sixth power of R. That means that in the case of the plates the attraction falls off much more slowly which can lead to significant dispersion forces even at relatively long separation.

This paper, while not leading to very novel or practical results, sheds light through detailed investigation of the possible role water can play between amyloid sheets. It again demonstrates the important effect that choice of methods and parameters can have on observations.

Krone, M.G., Hua, L., Soto, P., Zhou, R., Berne, B.J., Shea, J. (2008). Role of Water in Mediating the Assembly of Alzheimer Amyloid Aß (16-22) Protofilaments. Journal of the American Chemical Society, 130(33), 11066-11072. DOI: 10.1021/ja8017303