Large tilts in transmembrane helices can be induced during tertiary structure formation.

While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.

An introduction to membrane proteins.

α-Helical membrane proteins are important for many biological functions. Due to physicochemical constraints, the structures of membrane proteins differ from the structure of soluble proteins. Historically, membrane protein structures were assumed to be more or less two-dimensional, consisting of long, straight, membrane-spanning parallel helices packed against each other. However, during the past decade, a number of the new membrane protein structures cast doubt on this notion. Today, it is evident that the structures of many membrane proteins are equally complex as for many soluble proteins. Here, we review this development and discuss the consequences for our understanding of membrane protein biogenesis, folding, evolution, and bioinformatics.

Why are polar residues within the membrane core evolutionary conserved?

Here, we present a study of polar residues within the membrane core of alpha-helical membrane proteins. As expected, polar residues are less frequent in the membrane than expected. Further, most of these residues are buried within the interior of the protein and are only rarely exposed to lipids. However, the polar groups often border internal water filled cavities, even if the rest of the sidechain is buried. A survey of their functional roles in known structures showed that the polar residues are often directly involved in binding of small compounds, especially in channels and transporters, but other functions including proton transfer, catalysis, and selectivity have also been attributed to these proteins. Among the polar residues histidines often interact with prosthetic groups in photosynthetic- and oxidoreductase-related proteins, whereas prolines often are required for conformational changes of the proteins. Indeed, the polar residues in the membrane core are more conserved than other residues in the core, as well as more conserved than polar residues outside the membrane. The reason is twofold; they are often (i) buried in the interior of the protein and (ii) directly involved in the function of the proteins. Finally, a method to identify which polar residues are present within the membrane core directly from protein sequences was developed. Applying the method to the set of all human membrane proteins the prediction indicates that polar residues were most frequent among active transporter proteins and GPCRs, whereas infrequent in families with few transmembrane regions, such as non-GPCR receptors.

MPRAP: an accessibility predictor for a-helical transmembrane proteins that performs well inside and outside the membrane.

BACKGROUND: In water-soluble proteins it is energetically favorable to bury hydrophobic residues and to expose polar and charged residues. In contrast to water soluble proteins, transmembrane proteins face three distinct environments; a hydrophobic lipid environment inside the membrane, a hydrophilic water environment outside the membrane and an interface region rich in phospholipid head-groups. Therefore, it is energetically favorable for transmembrane proteins to expose different types of residues in the different regions. RESULTS: Investigations of a set of structurally determined transmembrane proteins showed that the composition of solvent exposed residues differs significantly inside and outside the membrane. In contrast, residues buried within the interior of a protein show a much smaller difference. However, in all regions exposed residues are less conserved than buried residues. Further, we found that current state-of-the-art predictors for surface area are optimized for one of the regions and perform badly in the other regions. To circumvent this limitation we developed a new predictor, MPRAP, that performs well in all regions. In addition, MPRAP performs better on complete membrane proteins than a combination of specialized predictors and acceptably on water-soluble proteins. A web-server of MPRAP is available at http://mprap.cbr.su.se/ CONCLUSION: By including complete a-helical transmembrane proteins in the training MPRAP is able to predict surface accessibility accurately both inside and outside the membrane. This predictor can aid in the prediction of 3D-structure, and in the identification of erroneous protein structures.

Membrane insertion of marginally hydrophobic transmembrane helices depends on sequence context.

In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.

Structure is three to ten times more conserved than sequence--a study of structural response in protein cores.

Protein structures change during evolution in response to mutations. Here, we analyze the mapping between sequence and structure in a set of structurally aligned protein domains. To avoid artifacts, we restricted our attention only to the core components of these structures. We found that on average, using different measures of structural change, protein cores evolve linearly with evolutionary distance (amino acid substitutions per site). This is true irrespective of which measure of structural change we used, whether RMSD or discrete structural descriptors for secondary structure, accessibility, or contacts. This linear response allows us to quantify the claim that structure is more conserved than sequence. Using structural alphabets of similar cardinality to the sequence alphabet, structural cores evolve three to ten times slower than sequences. Although we observed an average linear response, we found a wide variance. Different domain families varied fivefold in structural response to evolution. An attempt to categorically analyze this variance among subgroups by structural and functional category revealed only one statistically significant trend. This trend can be explained by the fact that beta-sheets change faster than alpha-helices, most likely due to that they are shorter and that change occurs at the ends of the secondary structure elements.

Coils in the membrane core are conserved and functionally important.

With the increasing number of available alpha-helical transmembrane (TM) protein structures, the traditional picture of membrane proteins has been challenged. For example, reentrant regions, which enter and exit the membrane at the same side, and interface helices, which lie parallel with the membrane in the membrane-water interface, are common. Furthermore, TM helices are frequently kinked, and their length and tilt angle vary. Here, we systematically analyze 7% of all residues within the deep membrane core that are in coil state. These coils can be found in TM-helix kinks as major breaks in TM helices and as parts of reentrant regions. Coil residues are significantly more conserved than other residues. Due to the polar character of the coil backbone, they are either buried or located near aqueous channels. Coil residues are frequently found within channels and transporters, where they introduce the flexibility and polarity required for transport across the membrane. Therefore, we believe that coil residues in the membrane core, while constituting a structural anomaly, are essential for the function of proteins.