Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.

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.

Positively charged residues located near the cytoplasmic end of hydrophobic segments in membrane proteins promote membrane insertion and formation of transmembrane ␣-helices. A quantitative understanding of this effect has been lacking, however. Here, using an in vitro transcription-translation system to study the insertion of model hydrophobic segments into dog pancreatic rough microsomes, we show that a single Lys or Arg residue typically contributes approximately ؊0.5 kcal/mol to the apparent free energy of membrane insertion (⌬G app) when placed near the cytoplasmic end of a hydrophobic segment and that stretches of 3-6 Lys residues can contribute significantly to ⌬Gapp from a distance of up to Ϸ13 residues away. membrane protein ͉ positive inside rule ͉ hydrophobicity scale ͉ translocon T he biosynthesis of ␣-helical membrane proteins requires their insertion, folding, and oligomerization in the target membrane. In eukaryotic cells, most membrane proteins insert cotranslationally into the endoplasmic reticulum (ER) membrane in a process mediated by the heterotrimeric Sec61 translocon (1, 2).In a series of recent studies (3-6), we have determined the sequence characteristics responsible for the insertion of a transmembrane helix into the ER membrane by measuring the membrane-insertion efficiency of designed hydrophobic segments (H-segments) engineered into a model protein. The position-dependent apparent free energy of insertion derived from these studies for the different amino acids provides the basis for a truly ''biological'' hydrophobicity scale (6). The biological scale correlates well with biophysical and statistical hydrophobicity scales, such as the Wimley-White water-octanol partitioning scale (7) and Sansom's structure-based statistical scale (8). This correlation suggests that the insertion of transmembrane helices is largely determined by the thermodynamics of protein-lipid interactions (3), which is not unreasonable given the structure of the archeal Sec61 translocon in which a dynamic ''lateral gate'' may provide a polypeptide in transit ready access to the lipid bilayer (9, 10).The biosynthesis of membrane proteins, however, not only requires the proper insertion of the transmembrane segments but also their correct orientation in the membrane, and the translocon is thought to play a pivotal role in this process as well (reviewed in ref. 11). Positively charged residues in membrane proteins are found predominantly on the cytoplasmic side of the membrane [the ''positive inside'' rule (12, 13)], flanking either signal sequences (14) or transmembrane segments (12). Consistent with this rule, positively charged residues favor insertion when placed at the cytoplasmic end of a transmembrane segment (6).To characterize more fully the effects on membrane insertion of charged flanking residues, we have now measured the apparent free energy of membrane insertion of a set of H-segments with different combinations of charged flanking residues engineered into a model membrane protein. We have tested the ef...

The minimal model system to study the basic principles of protein folding is the hairpin. The formation of beta-hairpins, which are the basic components of antiparallel beta-sheets, has been studied extensively in the past decade, but much less is known about helical hairpins. Here, we probe hairpin formation between a polyproline type-II helix and an alpha-helix as present in the natural miniprotein peptide YY (PYY). Both turn sequence and interactions of aromatic side chains from the C-terminal alpha-helix with the pockets formed by N-terminal Pro residues are shown by site-directed mutagenesis and solution NMR spectroscopy in different solvent systems to be important determinants of backbone dynamics and hairpin stability, suggesting a close analogy with some beta-hairpin structures. It is shown that multiple relatively weak contacts between the helices are necessary for the formation of the helical hairpin studied here, whereas the type-I beta-turn acts like a hinge, which through certain single amino acid substitutions is destabilized such that hairpin formation is completely abolished. Denaturation and renaturation of tertiary structure by temperature or cosolvents were probed by measuring changes of chemical shifts. Folding of PYY is both reversible and cooperative as inferred from the sigmoidal denaturation curves displayed by residues at the interface of the helical hairpin. Such miniproteins thus feature an important hallmark of globular proteins and should provide a convenient system to study basic aspects of helical hairpin folding that are complementary to those derived from studies of beta-hairpins.