The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports first-row transition metal ions Zn2+ and Co2+, but not Ca2+, across membranes. The conduction path was designed to contain two di-metal binding sites that bind with negative cooperativity. X-ray crystallography and solid-state and solution nuclear magnetic resonance indicate that the overall helical bundle is formed from two tightly interacting pairs of helices, which form individual domains that interact weakly along a more dynamic interface. Vesicle flux experiments show that as Zn2+ ions diffuse down their concentration gradients, protons are antiported. These experiments illustrate the feasibility of designing membrane proteins with predefined structural and dynamic properties.

Understanding the energetics of molecular interactions is fundamental to all of the central quests of structural biology including structure prediction and design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Hydrogen-bonding is widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water. Indeed, polar residue substitutions are the most common disease-causing mutations in membrane proteins. Because of limited structural information and technical challenges, however, there have been few quantitative tests of hydrogen-bond strength in the context of large membrane proteins. Here we show, by using a double-mutant cycle analysis, that the average contribution of eight interhelical side-chain hydrogen-bonding interactions throughout bacteriorhodopsin is only 0.6 kcal mol(-1). In agreement with these experiments, we find that 4% of polar atoms in the non-polar core regions of membrane proteins have no hydrogen-bond partner and the lengths of buried hydrogen bonds in soluble proteins and membrane protein transmembrane regions are statistically identical. Our results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing. Weak hydrogen-bonding should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.

The amino acid sequences of transmembrane regions of helical membrane proteins are highly constrained, diverging at slower rates than their extramembrane regions and than water-soluble proteins. Moreover, helical membrane proteins seem to fall into fewer families than water-soluble proteins. The reason for the differential restrictions on sequence remains unexplained. Here, we show that the evolution of transmembrane regions is slowed by a previously unrecognized structural constraint: Transmembrane regions bury more residues than extramembrane regions and soluble proteins. This fundamental feature of membrane protein structure is an important contributor to the differences in evolutionary rate and to an increased susceptibility of the transmembrane regions to disease-causing single-nucleotide polymorphisms.disease mutation ͉ potassium channel ͉ protein folding ͉ protein stability ͉ single nucleotide polymorphisms E volutionary rates vary considerably in different cellular compartments (1). Membrane proteins have been found to diverge faster overall than soluble proteins (2, 3), but this increased rate is confined entirely to the rapidly evolving extramembrane regions. Transmembrane regions, on average, diverge much more slowly than the extramembrane regions more slowly than soluble proteins (1, 4-6).A major factor controlling protein sequence divergence is the need to preserve protein function by maintaining a folded structure (7). Because the physical forces that drive folding can change with environment, proteins in different cellular locations can be subject to distinct evolutionary constraints. Membrane proteins, in particular, must accommodate to a dramatically varied environment, ranging from hydrocarbon chains in the bilayer core to water as they emerge from the membrane (8, 9). It therefore seems possible that distinct structural imperatives found in different environments could be an important contributor to evolutionary rates. An obvious sequence adaptation is the hydrophobic matching of the protein exterior, reflected in an apolar transmembrane amino acid composition. Although amino acid diversity is more limited in the transmembrane segments, simple compositional differences do not explain the slower divergence rates of transmembrane regions (1, 4, 5).Here, we find that the transmembrane regions of membrane proteins bury more residues on average than soluble proteins and much more than extramembrane regions, a possible mechanism for increasing stabilization in the absence of the hydrophobic effect. Because buried residues evolve at slower rates than surface residues (10-12), the higher level of residue burial in the transmembrane regions leads to slower sequence divergence. Moreover, we find that higher residue burial may explain a higher prevalence of disease-causing mutations in the transmembrane region of membrane proteins compared with the extramembrane regions. Results and DiscussionTransmembrane Regions Bury More Residues. Fig. 1A shows plots of the fractional surface area buried per residue v...

A major driving force for water soluble protein folding is the hydrophobic effect, but membrane proteins can not make use of this stabilizing contribution in the apolar core of the bilayer. It has been proposed that membrane proteins compensate by packing more efficiently. We therefore investigated packing contributions experimentally by observing the energetic and structural consequences of cavity creating mutations in the core of a membrane protein. We observed little difference in the packing energetics of water and membrane soluble proteins. Our results imply that other mechanisms are employed to stabilize the structure of membrane proteins.The hydrophobic effect is a major contributor to the stability of water soluble proteins, 1 but is essentially absent in the hydrocarbon core of a membrane. 2 Thus, the relative importance of other factors such as hydrogen bonding and van der Waals packing interactions must increase. While some hydrogen bonds can be key drivers of helix association, 3-5 we have argued that most appear to be modest contributors to tertiary structure stabilization in large membrane proteins. 2,6 If so, it implies that van der Waals packing dominates. Indeed, transmembrane helix dimers can be formed without any polar residues in the interface. 7 E-mail: bowie@mbi.ucla.edu. Supporting Information Available: Materials and methods, thermodynamic parameters for protein unfolding, X-ray diffraction data statistics, PDB codes for the mutant structures, and cavity measurements. This material is available free of charge via the Internet at http://pubs.acs.org. The lack of a clear consensus on packing density differences indicates that if they exist, it remains a rather subtle effect. NIH Public AccessGeometric analysis of static crystal structures is not always directly translatable to energetics, so we decided to probe packing contributions experimentally. Our analysis follows the classic work of Matthews and co-workers on the soluble protein T4 lysozyme. 12 In their work, the structural and energetic consequences of Leu-to-Ala substitutions in the protein core were investigated. The results are recapitulated in Fig. 1. They found a remarkably linear correlation between the change in thermodynamic stability and increased cavity size, in terms of both volume and surface area, created by the core substitutions. The extrapolated free energy at zero change in cavity size (1.9 kcal mol −1 ) provides an estimate of the change in the desolvation contribution alone (i.e. due to the hydrophobic effect), without a contribution from decreased packing. The slope of the lines (24 ± 3 cal mol −1 Å −3 and 20 ± 5 cal mol −1 Å −3 ) reflects the energetic cost of lost packing in the core of T4 lysozyme.To assess packing contributions in the core of a membrane protein, we made a set of large to small substitutions at buried residues in bacteriorhopsin (V49A, L94A, L111A, I148A, I148V and L152A) and obtained crystal structures for each of these mutants (see Table S1 in the Supporting Information). The most rel...

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