title: Coassembly of synthetic segments of shaker K+ channel within phospholipid membranes.
abstract: Increasing evidence suggests that membrane-embedded hydrophobic segments can interact within the phospholipid milieu of the membrane with varying degrees of specificity and thus contribute to the folding and oligomerization of proteins. We have used synthetic peptides corresponding to segments from the hydrophobic core of the Shaker potassium channel as a model system to study interactions between membrane-embedded segments. Three synthetic segments of the Shaker K+ channel, comprising the hydrophobic S2, S3, and S4 sequences, were used, and their secondary structure, their interactions with, and orientation within phospholipid membranes were examined. Secondary structure studies revealed that though S3 and S4 both adopt certain fractions of alpha-helical structures in membrane mimetic environments, the alpha-helical content of S3 is lower. Both S3 and S4 bind strongly to zwitterionic phospholipids, with partition coefficients in the order of 10(4) and 10(5) M-1. ATR-FTIR studies showed that while the S4 peptide is oriented parallel to the membrane surface, S3 tends to a more transmembranal orientation. Enzymatic cleavage experiments demonstrated that the presence of S3 induces some change in the proteolytic accessibility of the S4 segment. Resonance energy transfer measurements, done in high lipid/peptide molar ratios, revealed that S3 and S4 cannot self-associate in zwitterionic phospholipid vesicles but can associate with each other and with the S2 segment of the channel. Furthermore, S3 does not interact with the homologous S4 region from the first repeat of the eel sodium channel, demonstrating specificity in the interactions. These results are in line with data indicating that functionally important interactions indeed exist between the negatively charged S2 and S3 regions and the positively charged S4 region [Papazian, D. M., et al (1995) Neuron 14, 1293-1301; Planells-Cases, R., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 92, 9422-9426]. From a broader point of view, these results provide further support to the notion that interactions (either specific or nonspecific) may exist between transmembrane segments of integral membrane proteins and therefore can contribute to their assembly and organization.
authors.affiliation: Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: H
authors.initials: H
authors.lastname: Peled-Zehavi
authors.forename: I T
authors.initials: IT
authors.lastname: Arkin
authors.forename: D M
authors.initials: DM
authors.lastname: Engelman
authors.forename: Y
authors.initials: Y
authors.lastname: Shai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004735:Energy Transfer; D008081:Liposomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism*; D015221:Potassium Channels / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051637:Shaker Superfamily of Potassium Channels; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D008081:Liposomes; D010446:Peptide Fragments; D015221:Potassium Channels; D051637:Shaker Superfamily of Potassium Channels