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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.

SUBMITTER: Gratkowski H

PROVIDER: S-EPMC14678 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Polar side chains drive the association of model transmembrane peptides.

Gratkowski H H Lear J D JD DeGrado W F WF

Proceedings of the National Academy of Sciences of the United States of America 20010101 3

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 hydrophob ...[more]

PMID: 11158564

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Project description:The transition from the closed to open state greatly alters the intra- and inter-subunit interactions of the P2X receptor (P2XR). The interactions that occur in the transmembrane domain of the P2X2R remain unclear. We used substituted cysteine mutagenesis disulfide mapping to identify pairs of residues that are in close proximity within the transmembrane domain of rP2X2R and compared our results to the predicted positions of these amino acids obtained from a rat P2X2R homology model of the available open and closed zebrafish P2X4R structures. Alternations in channel function were measured as a change in the ATP-gated current before and after exposure to dithiothreitol. Thirty-six pairs of double mutants of rP2X2R expressed in HEK293 cells produced normal functioning channels. Thirty-five pairs of these mutants did not exhibit a functionally detectable disulfide bond. The double mutant H33C/S345C formed redox-dependent cross-links in the absence of ATP. Dithiothreitol ruptured the disulfide bond of H33C/S345C and induced a 2 to 3-fold increase in current. The EC50 for H33C/S345C before dithiothreitol treatment was ~2-fold higher than that after dithiothreitol treatment. Dithiothreitol reduced the EC50 to wild-type levels. Furthermore, expression of trimeric concatamer receptors with Cys mutations at some but not all six positions showed that the more disulfide bond formation sites within the concatamer, the greater current potentiation after dithiothreitol incubation. Immunoblot analysis of H33C/S345C revealed one monomer band under nonreducing conditions strongly suggesting that disulfide bonds are formed within single subunits (intra-subunit) and not between two subunits (inter-subunit). Taken together, these data indicate that His33 and Ser345 are proximal to each other across an intra-subunit interface. The relative movement between the first transmembrane and the second transmembrane in the intra-subunit is likely important for transmitting the action of ATP binding to the opening of the channel.

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Polar side chains drive the association of model transmembrane peptides.

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Polar side chains drive the association of model transmembrane peptides.

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