Hydrophobic helical hairpins: design and packing interactions in membrane environments.
Helix-helix interactions within membranes are dominated by van der Waals packing motifs and side chain-side chain hydrogen bond formation, which act in tandem to determine the residues that comprise the interface between two given helices. To explore in a systematic manner the tertiary contacts between transmembrane helices, we have designed and expressed in Escherichia coli highly hydrophobic helix-loop-helix constructs of prototypic sequence K(1)KKKKKKFAIAIAIIAWAX(19)AIIAIAIAIKSPGSKIAIAIAIIAZ(44)AWAIIAIAIAFKKKKKKK(62), where "small" (Ala) and "large" (Ile) residues were used to maximize the tertiary contact area. Evidence that the two transmembrane (TM) segments in the AI construct contain an interface conducive for folding into a hairpin structure was obtained from the results that (i) the single TM AI(pep) peptide derived from the AI hairpin forms SDS-resistant dimers on PAGE gels and (ii) the corresponding sequence forms a strong dimer when examined in vivo in TOXCAT assays. Site-directed mutagenesis of AI hairpins was carried out to incorporate each of the 20 commonly occurring amino acids at X positions. Analysis on Western blots using an oligomerization assay in 12% NuPage-sodium dodecyl sulfate (SDS) indicated that mutants with X = E, D, Q, R, N, H, and K largely formed SDS-resistant dimers-which likely correspond to H-bonded four-helix bundles-while all the others (e.g., X = F, W, L, I, M, V, C, Y, A, T, S, G, and P) remained monomeric. Systematic studies of X/Z double mutants indicated that formation of hairpin dimers is the result of the disruption of stabilizing interactions between the antiparallel helices within the AI construct. The overall results suggest that, in situations where hydrophobic van der Waals packing energy between helices is sufficient to prevent significant rotation about the major axes of interacting helices, intrahairpin side chain-side chain H-bond formation will occur mainly when pairs of polar residues are interfacially located and proximal. Knowledge of the relative contributions of these forces should be of value, for example, in clarifying the context--and the structural consequences--of disease-related mutations.
Johnson
Rachel M
RM
Division of Structural Biology and Biochemistry, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Heslop
Claire L
CL
Deber
Charles M
CM
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005822:Genetic Vectors; D005973:Glutamine / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism*; D008823:Micelles; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary / Q000235:genetics
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008823:Micelles; D005973:Glutamine; D001224:Aspartic Acid