The forces stabilizing the three-dimensional structures of membrane proteins are currently not well understood. Previously, it was shown that a single Asn side chain in a transmembrane segment can mediate the dimerization and trimerization of a variety of hydrophobic helices. Here, we examine the tendencies of a representative set of amino acids (Asn, Gln, Asp, Glu, Lys, Ala, Val, Leu, Ser, Thr) to direct the oligomerization of a model transmembrane helix. The model peptide is entirely hydrophobic throughout a 20-residue segment and contains a single central site for the introduction of various amino acid ''guests.'' Analytical ultracentrifugation and gel electrophoresis were used to determine the stoichiometry and free energy of association of the entire set of peptides within micelles. Variants with two polar atoms at the guest site-Asn, Gln, Asp, and Glu-formed stable trimers, whereas residues with one or fewer polar atoms showed a much weaker tendency to associate. The data are examined in light of the frequencies of occurrence of various amino acid side chains in membrane proteins and provide insight into the role of polar interactions in directing transmembrane helix association. These data also suggest an approach to the design of variants of natural single-span transmembrane proteins with various potentials to associate in the bilayer. T he forces that drive membrane protein folding are not well understood. A reasonable model for membrane protein folding (1) posits that folding occurs in two kinetically separate steps. The first involves insertion of the helical regions of the protein in the bilayer, whereas the second involves the formation of specific interactions between these helices, to form a tightly packed native structure. The second step can occur in an intermolecular process as in the folding͞assembly of multimeric ion channel proteins or in an intramolecular process, as in the folding of monomolecular proteins. The features required for the insertion of peptides into bilayers are largely hydrophobic in nature and have been quantified through various model systems (reviewed in ref. 2). However, the features required for the subsequent association of inserted helices are controversial and less well understood (3). One early study (4) suggested that the composition of the interiors of membrane proteins are similar to those of water-soluble proteins; both predominantly consist of well-packed apolar residues. Buried polar side chains occur less frequently but may be important for function, conformational specificity, and thermodynamic stability. A more recent study suggests that small residues such as Gly and Ala may additionally play an important role in the association of some transmembrane helix pairs (5-7), although this is not a universal phenomenon (8). Finally, the role of amino acid sequence in defining the orientation and topology of transmembrane helical segments has been defined by using a glycosylation mapping method (9-11).To determine experimentally the features required for folding m...

Transmembrane helices of integrin alpha and beta subunits have been implicated in the regulation of integrin activity. Two mutations, glycine-708 to asparagine-708 (G708N)and methionine-701 to asparagine-701, in the transmembrane helix of the beta3 subunit enabled integrin alphaIIbbeta3 to constitutively bind soluble fibrinogen. Further characterization of the G708N mutant revealed that it induced alphaIIbbeta3 clustering and constitutive phosphorylation of focal adhesion kinase. This mutation also enhanced the tendency of the transmembrane helix to form homotrimers. These results suggest that homomeric associations involving transmembrane domains provide a driving force for integrin activation. They also suggest a structural basis for the coincidence of integrin activation and clustering.

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic ␣-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly 15 N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane ␣-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu 2-51 (residues 2-51), which contains the transmembrane ␣-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu 28 -81 (residues 28 -81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2-81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ؎ 3 pS and 12 ؎ 3 pS in 0.5 M KCl and 29 ؎ 3 pS and 12 ؎ 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu 2-51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu 28 -81, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.