Polar side chains drive the association of model transmembrane peptides

Abstract

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...

Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein

“…The inclusion of glutamine at position 17 and aspartic acid at position 33 in the library likely biased the interactions toward the PDGF b receptor and accounts for the ability of such a high percentage of the library proteins to transform cells. This result is consistent with biochemical studies which showed that insertion of hydrophilic residues into monomeric hydrophobic peptides induced them to homodimerize with high affinity by forming interhelical hydrogen bonds, but the strength of such hydrophilic interactions may facilitate the formation of nonspecific transmembrane complexes as well (Zhou et al, 2000;Gratkowski et al, 2001).…”

“…Most amino acids in transmembrane domains are hydrophobic, and specific interhelical interactions can be mediated by van der Waals contacts between these hydrophobic side chains (Lemmon et al, 1992b(Lemmon et al, , 1994Bowie, 1997;Fleming et al, 1997;Langosch and Heringa, 1998;Gurezka et al, 1999;Eilers et al, 2000;Russ and Engelman, 2000;Adamian and Liang, 2001). Hydrophilic residues can also contribute strong interactions to the oligomerization of some transmembrane helices through hydrogen bond formation or charge pair interactions (e.g., Manolios et al, 1990;Cosson et al, 1991;Smith et al, 1996;Choma et al, 2000;Zhou et al, 2000;Gratkowski et al, 2001;Senes et al, 2001;Therien et al, 2001;Dawson et al, 2002;Partridge et al, 2002).…”

Sequence-specific Dimerization of the Transmembrane Domain of the “BH3-only” Protein BNIP3 in Membranes and Detergent

“…We targeted position His 173 within the TM domain because of previous reports (27)(28)(29)(30) that polar side chains can drive helix-helix association. Because mutations at His 173 have graded effects on dimerization in both TOXCAT and SDS-PAGE assays, our results demonstrate that this polar residue does help to stabilize TM domain interactions.…”

“…Strongly polar residues within hydrophobic TM domains have been shown to drive oligomerization in membranes and detergents (27)(28)(29)(30), and the GXXXG motif has been shown to mediate interactions between TMs in several biological systems (31-37). Mutational analysis shows that the TM domain interaction is mediated by both polar interactions and the motif AXXXG, and that the detergentsolubilized dimer is disrupted by the same mutations that disrupt the membrane-embedded complex.…”

Structural differences between thermophilic and mesophilic membrane proteins

“…[8][9][10][11][12] Although most hydrogen-bonded sidechain interactions that have been experimentally measured in membrane proteins appear to make relatively modest contributions, [21][22][23][24][25][26][27] some have been measured as high as 1.8 kcal/mol and in theory it is possible that they could be made even stronger. 26,28 Thus, it seems reasonable to expect that the number of transmembrane interhelical hydrogen bonds might increase in thermophiles.…”