Polar and ionizable amino acid residues are frequently found in the transmembrane (TM) regions of membrane proteins. In this study, we show that they help to form extensive hydrogen bond connections between TM helices. We find that almost all TM helices have interhelical hydrogen bonding. In addition, we find that a pair of contacting TM helices is packed tighter when there are interhelical hydrogen bonds between them. We further describe several spatial motifs in the TM regions, including "Polar Clamp" and "Serine Zipper," where conserved Ser residues coincide with tightly packed locations in the TM region. With the examples of halorhodopsin, calcium-transporting ATPase, and bovine cytochrome c oxidase, we discuss the roles of hydrogen bonds in stabilizing helical bundles in polytopic membrane proteins and in protein functions.
Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (TransMembrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of high-resolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 A. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis.
Recent studies with model peptides and statistical analyses of the crystal structures of membrane proteins have shown that buried polar interactions contribute significantly to the stabilization of the three-dimensional structures of membrane proteins. Here, we probe how the location of these polar groups along the transmembrane helices affect their free energies of interaction. Asn residues were placed singly and in pairs at three positions within a model transmembrane helix, which had previously been shown to support the formation of trimers in micelles. The model helix was designed to form a transmembrane coiled coil, with Val side chains at the "a" positions of the heptad repeat. Variants of this peptide were prepared in which an Asn residue was introduced at one or more of the "a" positions, and their free energies of association were determined by analytical ultracentrifugation. When placed near the middle of the transmembrane helix, the formation of trimers was stabilized by at least -2.0 kcal/mol per Asn side chain. When the Asn was placed at the interface between the hydrophobic and polar regions of the peptide, the substitution was neither stabilizing nor destabilizing (0.0 +/- 0.5 kcal/mol of monomer). Finally, it has previously been shown that a Val-for-Asn mutation in a water-soluble coiled coil destabilizes the structure by approximately 1.5 kcal/mol of monomer [Acharya, A., et al. (2002) Biochemistry 41, 14122-14131]. Thus, the headgroup region of a micelle appears to have a conformational impact intermediate between that of bulk water and the apolar region of micelle. A similarly large dependence on the location of the polar residues was found in a statistical survey of helical transmembrane proteins. The tendency of different types of residues to be buried in the interiors versus being exposed to lipids was analyzed. Asn and Gln show a very strong tendency to be buried when they are located near the middle of a transmembrane helix. However, when placed near the ends of transmembrane helices, they show little preference for the surface versus the interior of the protein. These data show that Asn side chains within the apolar region of the transmembrane helix provide a significantly larger driving force for association than Asn residues near the apolar/polar interface. Thus, although polar interactions are able to strongly stabilize the folding of membrane proteins, the energetics of association depend on their location within the hydrophobic region of a transmembrane helix.
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