Daisuke Noshiro

Extramembrane Control of Ion Channel Peptide Assemblies, Using Alamethicin as an Example

Ion channels allow the influx and efflux of specific ions through a plasma membrane. Many ion channels can sense, for example, the membrane potential (the voltage gaps between the inside and the outside of the membrane), specific ligands such as neurotransmitters, and mechanical tension within the membrane. They modulate cell function in response to these stimuli. Researchers have focused on developing peptide- and non-peptide-based model systems to elucidate ion-channel protein functions and to create artificial sensing systems. In this Account, we employed a typical peptide that forms ion channels,alamethicin, as a model to evaluate our methodologies for controlling the assembly states of channel-forming molecules in membranes. As alamethicin self-assembles in membranes, it prompts channel formation, but number of peptide molecules in these channels is not constant. Using planar-lipid bilayer methods, we monitored the association states of alamethicin in real time. Many ligand-gated, natural-ion channel proteins have large extramembrane domains. As these proteins interact with specific ligands, those conformational alterations in the extramembrane domains are transmitted to the transmembrane, pore-forming domains to open and close the channels. We hypothesized that if we conjugated suitable extramembrane segments to alamethicin, ligand binding to the extramembrane segments could alter the structure of the extramembrane domains and influence the association states or association numbers of alamethicin in the membranes. We could then assess those changes by using single-channel current recording. We found that we could modulate channel assembly and eventual ion flux with attached leucine-zipper extramembrane peptide segments. Using conformationally switchable leucine-zipper extramembrane segments that respond to Fe(3+), we fabricated an artificial Fe(3+)-sensitive ion channel; a decrease in the helical content of the extramembrane segment led to an increase in the channel current. When we added a calmodulin C-terminus segment, we formed a channel that was sensitive to Ca(2+). This result demonstrated that we could prepare artificial channels that were sensitive to specific ligands by adding appropriate extramembrane segments from natural protein motifs that respond to external stimuli. In conclusion, our research points to the possibility of creating tailored sensor or signal transduction systems through the conjugation of a conformationally switchable extramembrane peptide/protein segment to a suitable transmembrane peptide segment.

Metal-Assisted Channel Stabilization: Disposition of a Single Histidine on the N-terminus of Alamethicin Yields Channels with Extraordinarily Long Lifetimes

Alamethicin, a member of the peptaibol family of antibiotics, is a typical channel-forming peptide with a helical structure. The self-assembly of the peptide in the membranes yields voltage-dependent channels. In this study, three alamethicin analogs possessing a charged residue (His, Lys, or Glu) on their N-termini were designed with the expectation of stabilizing the transmembrane structure. A slight elongation of channel lifetime was observed for the Lys and Glu analogs. On the other hand, extensive stabilization of certain channel open states was observed for the His analog. This stabilization was predominantly observed in the presence of metal ions such as Zn(2+), suggesting that metal coordination with His facilitates the formation of a supramolecular assembly in the membranes. Channel stability was greatly diminished by acetylation of the N-terminal amino group, indicating that the N-terminal amino group also plays an important role in metal coordination.

Metal‐Stimulated Regulation of Transcription by an Artificial Zinc‐Finger Protein

Regulation of the expression of specific genes at a desired time opens attractive avenues for research in chemical biology, cell biology, and future gene therapy. A C2H2-type zinc-finger motif, one of the most ubiquitous DNA binding motifs, binds a zinc ion through two conserved cysteine and two histidine residues. Zn binding is necessary for the proper folding of the peptides into a globular bba structure and for the DNA binding ability. The binding between a typical C2H2-type zincfinger peptide and Zn is extremely stable. Such C2H2-type zinc fingers always preserve the Zn ions inside the cells, and thus, it is difficult to control their DNA binding ability by changing the Zn concentration. By substituting the zinc-ligating residue(s) of a C2H2-type zinc-finger motif, we expected that the binding affinity to Zn would decrease and that it would then be possible to control the DNA binding ability of the peptide through changes in the Zn concentration (Figure 1 A). Though Cys and His are important ligands for the formation of the zinc-finger structure, histidine residues are directly involved in the formation of the a-helix that is responsible for DNA recognition. Considering that the DNA binding function should not be disturbed by mutations, a Cys mutation was employed. According to the statistics of protein residues serving as zinc ligands, the usage of aspartic acid and glutamic acid follows that of Cys and His. We selected Asp as a substitute for Cys because of the similar side-chain lengths. The DNA binding domain of the wild-type Zif268 (wtZF3) consists of three C2H2-type zinc-finger motifs connected in tandem that bind to double-stranded DNA containing the 5’GCGTGGGCGT-3’ sequence (Zif binding sequence: ZBS). In order to examine the applicability of the above concept, a zinc-finger protein (CDH2-ZF3) was designed by substituting the second zinc-finger motif of wtZF3 into the CDH2-type (Figure 1). The DNA binding affinity was determined by electrophoretic mobility shift assays (EMSA) in the presence of 10 mm ZnSO4 and an additional 100 mm ethylenediaminetetraacetic acid (EDTA). In the presence of the ZnSO4, CDH2-ZF3 showed a DNA binding pattern similar to that of wtZF3 (Figure 2 A and B, top). The Kd value of CDH2-ZF3 was calculated to be 2.1 1.4 nm, which is only about four times lower than that of wtZF3 (Kd = 0.49 0.09 nm ; Table 1). Therefore, the ligand substitution of the second finger of wtZF3 has no substantial effect on the DNA binding affinity in the presence of Zn. As shown in Figure 2 A and B (bottom), the addition of EDTA dramatically decreased the DNA binding affinity of CDH2-ZF3

Control of leakage activities of alamethicin analogs by metals: Side chain-dependent adverse gating response to Zn2+

Alamethicin (Alm), an antimicrobial peptide rich in α-aminoisobutyric acid (Aib), is known to self-assemble to form channels in the membranes. Previously, we reported that HG-Alm, an Alm analog with a single His residue at the N-terminus, forms channel assemblies with extremely long lifetimes in the presence of Zn(2+). In this study, HG-Alm analogs, in the sequences of which all Aib residues were substituted by Leu, norvaline (Nva), or norleucine (Nle), were synthesized and their leakage activities were measured using fluorescent dye-loaded liposomes. We found that these peptides could be categorized into two classes with different gating responses to Zn(2+).

Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

A double-ring-shaped tetradecameric GroEL complex assists proper protein folding in cooperation with the cochaperonin GroES. The dynamic GroEL–GroES interaction reflects the allosteric intra- and inter-ring communications and the chaperonin reaction. Therefore, revealing this dynamic interaction is essential to understanding the allosteric communications and the operation mechanism of GroEL. Nevertheless, how this interaction proceeds in the chaperonin cycle has long been controversial. Here, we directly image the dynamic GroEL–GroES interaction under conditions with and without foldable substrate protein using high-speed atomic force microscopy. Then, the imaging results obtained under these conditions and our previous results in the presence of unfoldable substrate are compared. The molecular movies reveal that the entire reaction pathway is highly complicated but basically identical irrespective of the substrate condition. A prominent (but moderate) difference is in the population distribution of intermediate species: symmetric GroEL : GroES2 and asymmetric GroEL : GroES1 complexes, and GroES–unbound GroEL. This difference is mainly attributed to the longer lifetime of GroEL : GroES1 complexes in the presence of foldable substrate. Moreover, the inter-ring communication, which is the basis for the alternating action of the two rings, occurs at two distinct (GroES association and dissociation) steps in the main reaction pathway, irrespective of the substrate condition. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.

Construction of a Ca(2+)-gated artificial channel by fusing alamethicin with a calmodulin-derived extramembrane segment.

Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.

Calmodulin EF-hand peptides as Ca2+-switchable recognition tags

Calmodulin is a representative calcium‐binding protein comprised of four Ca2+‐binding motifs with a helix‐loop‐helix structure (EF‐hands). In this study, we clarified the potential of peptide segments derived from the third and fourth EF‐hands (EF3 and EF4) to act as recognition tags. Through an analysis of the mode of disulfide formation among cysteines inserted at the N‐ or C‐terminus of these peptide segments, EF3 and EF4 peptides were suggested to form a heterodimer with a topology similar to that in the wild‐type protein. Heterodimer formation was shown to be a function of the Ca2+ concentration, suggesting that these structures may be used as Ca2+‐switchable recognition tags. An example of an “EF‐tag” system involving the membrane fusion of liposomes decorated with EF3 and EF4 peptides is presented.

Calmodulin EF-hand peptides as Ca2+-switchable recognition tags: Calmodulin EF-Hand Peptides

Calmodulin is a representative calcium‐binding protein comprised of four Ca2+‐binding motifs with a helix‐loop‐helix structure (EF‐hands). In this study, we clarified the potential of peptide segments derived from the third and fourth EF‐hands (EF3 and EF4) to act as recognition tags. Through an analysis of the mode of disulfide formation among cysteines inserted at the N‐ or C‐terminus of these peptide segments, EF3 and EF4 peptides were suggested to form a heterodimer with a topology similar to that in the wild‐type protein. Heterodimer formation was shown to be a function of the Ca2+ concentration, suggesting that these structures may be used as Ca2+‐switchable recognition tags. An example of an “EF‐tag” system involving the membrane fusion of liposomes decorated with EF3 and EF4 peptides is presented.

Use of calmodulin EF-hand peptides as Ca(2+) -switchable recognition tags.

Calmodulin is a representative calcium‐binding protein comprised of four Ca2+‐binding motifs with a helix‐loop‐helix structure (EF‐hands). In this study, we clarified the potential of peptide segments derived from the third and fourth EF‐hands (EF3 and EF4) to act as recognition tags. Through an analysis of the mode of disulfide formation among cysteines inserted at the N‐ or C‐terminus of these peptide segments, EF3 and EF4 peptides were suggested to form a heterodimer with a topology similar to that in the wild‐type protein. Heterodimer formation was shown to be a function of the Ca2+ concentration, suggesting that these structures may be used as Ca2+‐switchable recognition tags. An example of an “EF‐tag” system involving the membrane fusion of liposomes decorated with EF3 and EF4 peptides is presented.