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PH responsiveness of fibrous assemblies of repeat-sequence amphipathic ?-helix polypeptides.

ABSTRACT: We reported previously that our designed polypeptide ?3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)3, forms not only an amphipathic ?-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its ?-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of ?3 were replaced with Ser. We prepared the following polypeptides: 2S?3 (LSTLAKA)3, in which all Glu residues were replaced with Ser residues; 6S?3 (LETLASA)3, in which all Lys residues were replaced with Ser; and 2S6S?3 (LSTLASA)3; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2S?3 formed an ?-helix under basic conditions and 6S?3 formed an ?-helix under acid conditions. In 1M KCl, they both formed ?-helices under a wide pH range. In addition, 2S?3 and 6S?3 formed fibrous assemblies under the same buffer conditions in which they formed ?-helices. ?-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6S?3 formed an ?-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in ?-helix formation.

SUBMITTER: Takei T

PROVIDER: S-EPMC4420536 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides.

Takei Toshiaki T Tsumoto Kouhei K Okonogi Atsuhito A Kimura Akiko A Kojima Shuichi S Yazaki Kazumori K Takei Tsunetomo T Ueda Takuya T Miura Kin-ichiro K

Protein science : a publication of the Protein Society 20150402 5

We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)3, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following poly ...[more]

PMID: 25694229

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Project description:BackgroundIntragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, Mollicutes. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins.ResultsUsing a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the Mollicutes. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by HGT and intra-genomic shuffling.ConclusionsWe describe novel features of PARCELs (Palindromic Amphipathic Repeat Coding ELements), a set of widely distributed repeat protein domains and coding sequences that were likely acquired through HGT by diverse unicellular microbes, further mobilized and diversified within genomes, and co-opted for expression in the membrane proteome of some taxa. Disseminated by multiple gene-centric vehicles, ORFs harboring these elements enhance accessory gene pools as part of the "mobilome" connecting genomes of various clades, in taxa sharing common niches.

Project description:One of the fundamental features of biomembranes is the ability to fuse or to separate. These processes called respectively membrane fusion and fission are central in the homeostasis of events such as those related to intracellular membrane traffic. Proteins that contain amphipathic helices (AHs) were suggested to mediate membrane fission via shallow insertion of these helices into the lipid bilayer. Here we analyze the AH-containing proteins that have been identified as essential for membrane fission and categorize them in few subfamilies, including small GTPases, Atg proteins, and proteins containing either the ENTH/ANTH- or the BAR-domain. AH-containing fission-inducing proteins may require cofactors such as additional proteins (e.g., lipid-modifying enzymes), or lipids (e.g., phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidic acid [PA], or cardiolipin). Both PA and cardiolipin possess a cone shape and a negative charge (-2) that favor the recruitment of the AHs of fission-inducing proteins. Instead, PtdIns(4,5)P2 is characterized by an high negative charge able to recruit basic residues of the AHs of fission-inducing proteins. Here we propose that the AHs of fission-inducing proteins contain sequence motifs that bind lipid cofactors; accordingly (K/R/H)(K/R/H)xx(K/R/H) is a PtdIns(4,5)P2-binding motif, (K/R)x6(F/Y) is a cardiolipin-binding motif, whereas KxK is a PA-binding motif. Following our analysis, we show that the AHs of many fission-inducing proteins possess five properties: (a) at least three basic residues on the hydrophilic side, (b) ability to oligomerize, (c) optimal (shallow) depth of insertion into the membrane, (d) positive cooperativity in membrane curvature generation, and (e) specific interaction with one of the lipids mentioned above. These lipid cofactors favor correct conformation, oligomeric state and optimal insertion depth. The most abundant lipid in a given organelle possessing high negative charge (more negative than -1) is usually the lipid cofactor in the fission event. Interestingly, naturally occurring mutations have been reported in AH-containing fission-inducing proteins and related to diseases such as centronuclear myopathy (amphiphysin 2), Charcot-Marie-Tooth disease (GDAP1), Parkinson's disease (?-synuclein). These findings add to the interest of the membrane fission process whose complete understanding will be instrumental for the elucidation of the pathogenesis of diseases involving mutations in the protein AHs.

Dataset Information

PH responsiveness of fibrous assemblies of repeat-sequence amphipathic ?-helix polypeptides.

Publications

pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides.

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