Abstract:
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Johnson RM,Heslop CL,Deber CMdoi
10.1021/bi0492760subject
Has Abstractpub_date
2004-11-16 00:00:00pages
14361-9issue
45eissn
0006-2960issn
1520-4995journal_volume
43pub_type
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