Target-selective one-way membrane fusion system based on a pH-responsive coiled coil assembly at the interface of liposomal vesicles

The coiled coil trimer structure is a common motif observed in membrane fusion processes of specific fusion proteins such as the hemagglutinin glycoprotein. The HA2 subunit in the hemagglutinin changes its conformation or geometry to be favorable to membrane fusion in response to endosomal weakly acidic pH. This pH responsiveness is indispensable to an artificial polypeptide-triggered delivery system as well as the membrane fusion reaction in biology. In this study, we have constructed an AAB-type coiled coil heteroassembled system that is sensitive to weakly acidic pH. The heterotrimer is formed from two kinds of polypeptides containing an Ala or a Trp residue at a hydrophobic a position, and it was observed that the Glu residue at the other a position induced an acidic pH-dependent conformational change. On the basis of this pH-responsive coiled coil heteroassembled system, a boronic acid coupled working polypeptide for the combination of an intervesicular complex with a sugarlike compound on the surface of the target liposome, and a supporting polypeptide for the construction of a pH-responsive heterotrimer with the working polypeptide were designed and synthesized. The process of membrane fusion was characterized by lipid-mixing, inner-leaflet lipid-mixing, and content-mixing assays. The target selective vesicle fusion is clearly observed at a weakly acidic pH, where the working polypeptides form a heterotrimeric coiled coil with the supporting polypeptides in a 1:2 binding stoichiometry and the surfaces between pilot and target vesicles come into close proximity to each other.     

Design and characterization of endosomal-pH-responsive coiled coils for constructing an artificial membrane fusion system

A weakly acidic pH-responsive polypeptide is believed to have the potential for an endosome escape function in a polypeptide-triggered delivery system. For constructing a membrane fusion device with pH-responsiveness, we have designed novel polypeptides that are capable of forming an α2 coiled coil structure. Circular dichroism spectroscopy reveals that a polypeptide, AP-LZ(EH5), with a Glu and His salt-bridge pair at a staggered position in the hydrophobic core forms a stable coiled coil structure only at endosomal pH values (pH 5.0 to 5.5). On the basis of their endosomal-pH responsiveness, a boronic acid/polypeptide conjugate (BA-H5-St) was also designed as a pilot molecule to construct a pH-responsive, one-way membrane fusion system with a sugarlike compound (phosphatidylinositol: PI)-containing liposome as a target. Membrane fusion behavior was characterized by lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays. These studies reveal that membrane fusion is clearly observed when the pH of the experimental system is changed from 7.4 (physiological condition) to 5.0 (endosomal condition).     

Highly sensitive and quantitative analysis of polyeptides using a new gradient system based on an abrupt change in adsorption of polypeptide to the reversed-phase column packing

During a study of 100 microL aliquots of urocortin containing various acetonitrile contents, we hypothesized that a change in the acetonitrile content in the solution across a specific content of acetonitrile (critical threshold) causes an abrupt change in adsorption capacity to the column packing. Circular dichroism measurements suggest that the conformational change induced by acetonitrile in the solution causes the abrupt change in adsorption capacity, and this solvent-induced conformational change is reversible across the critical threshold. This hypothesis can apply to various polypeptides with molecular weights range from 1007 to 6789 and to other organic solvents. A new gradient system utilizing the instant recovery of the adsorption capacity across the critical threshold was designed, and applied to the analysis of a 100 microL aliquot of various polypeptide solutions. The results suggest that use of a solution containing organic solvents more than the critical threshold allows successful dilution of polypeptides up to picomolar concentration range without any loss due to its adsorption during handling procedures and loading onto the LC system, and that a new gradient system enables quantitative analysis of polypeptides at picomolar concentrations in such solutions.     

Design, construction, and characterization of high-performance membrane fusion devices with target-selectivity

Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembrane-peptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and inner-leaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic α-helix formation possible as expected. The target-selective liposome-liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system.     

Cell Surface Assembly of HIV gp41 Six-Helix Bundles for Facile, Quantitative Measurements of Hetero-oligomeric Interactions

Helix-helix interactions are fundamental to many biological signals and systems and are found in homo- or heteromultimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six-helix bundles (6HBs) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Using a yeast surface two-hybrid (YS2H) system, a platform designed to detect protein-protein interactions occurring through a secretory pathway, we reconstituted 6HB complexes on the yeast surface, quantitatively measured the equilibrium and kinetic constants of soluble 6HB, and delineated the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for the facile characterization and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils.     

Metal-mediated peptide assembly: use of metal coordination to change the oligomerization state of an alpha-helical coiled-coil

Metal coordination is used to alter the oligomerization state of a designed peptide structure. The 30-residue polypeptide AQ-Pal14Pal21contains two metal-binding 4-pyridylalanine (Pal) residues on its solvent-exposed surface and exists as a very stable two-stranded alpha-helical coiled-coil. Upon the addition of Pt(en)(NO3)2, a significant conformational change to a metal-bridged, four-helix bundle is seen.     

SNARE derived peptide mimic inducing membrane fusion

SNARE proteins mediate membrane fusion between synaptic vesicles and the plasma membrane. A minimized peptide SNARE model system with reduced complexity was introduced combining the native SNARE transmembrane (TMD) and linker domains with artificial coiled-coil forming peptides. Specific membrane fusion initiated by coiled-coil recognition was shown by lipid and content mixing vesicle assays.     

Membrane binding and structure of de novo designed alpha-helical cationic coiled-coil-forming peptides.

We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide-membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level.     

Metal-ion-dependent GFP emission in vivo by combining a circularly permutated green fluorescent protein with an engineered metal-ion-binding coiled-coil

Coordination of metal ions significantly contributes to protein structures and functions. Here we constructed a fusion protein, consisting of a de novo designed, metal-ion-binding, trimeric coiled-coil and a circularly permutated green fluorescent protein (cpGFP), where the fluorescent emission from cpGFP was induced by metal ion coordination to the coiled-coil. A circularly permutated GFP, (191)cpGFP(190), was constructed by connecting the original N- and C-termini of GFP(UV) by a GGSGG linker and cleaving it between Asp(190) and Gly(191). The metal-ion-binding coiled-coil, IZ-HH, was designed to have three alpha-helical structures, with 12 His residues in the hydrophobic core of the coiled-coil structure. IZ-HH exhibited an unfolded structure, whereas it formed the trimeric coiled-coil structure in the presence of divalent metal ions, such as Cu(2+), Ni(2+), or Zn(2+). The fusion protein (191)cpGFP(190)-IZ-HH was constructed, in which (191)cpGFP(190) was inserted between the second and third alpha-helices of IZ-HH. Escherichia coli cells, expressing (191)cpGFP(190)-IZ-HH, exhibited strong fluorescence when the Cu(2+) and Zn(2+) ions were present in the medium, indicating that they passed through the cell membrane and induced the proper folding of the (191)cpGFP(190) domain. This strategy, in which protein function is regulated by a metal-ion-responsive coiled-coil, should be applicable to the design of various metal-ion-responsive, nonnatural proteins that work both in vitro and in vivo.     

Incorporating electron-transfer functionality into synthetic metalloproteins from the bottom-up

The alpha-helical coiled-coil motif serves as a robust scaffold for incorporating electron-transfer (ET) functionality into synthetic metalloproteins. These structures consist of a supercoiling of two or more aplha helices that are formed by the self-assembly of individual polypeptide chains whose sequences contain a repeating pattern of hydrophobic and hydrophilic residues. Early work from our group attached abiotic Ru-based redox sites to the most surface-exposed positions of two stranded coiled-coils and used electron-pulse radiolysis to study both intra- and intermolecular ET reactions in these systems. Later work used smaller metallopeptides to investigate the effects of conformational gating within electrostatic peptide-protein complexes. We have recently designed the C16C19-GGY peptide, which contains Cys residues located at both the "a" and "d" positions of its third heptad repeat in order to construct a nativelike metal-binding domain within its hydrophobic core. It was shown that the binding of both Cd(II) and Cu(I) ions induces the peptide to undergo a conformational change from a disordered random coil to a metal-bridged coiled-coil. However, whereas the Cd(II)-protein exists as a two-stranded coiled-coil, the Cu(I) derivative exists as a four-stranded coiled-coil. Upon the incorporation of other metal ions, metal-bridged peptide dimers, tetramers, and hexamers are formed. The Cu(I)-protein is of particular interest because it exhibits a long-lived (microsecond) room-temperature luminescence at 600 nm. The luminophore in this protein is thought to be a multinuclear CuI4Cys4(N/O)4 cage complex, which can be quenched by exogenous electron acceptors in solution, as shown by emission-lifetime and transient-absorption experiments. It is anticipated that further investigation into these systems will contribute to the expanding effort of bioinorganic chemists to prepare new kinds of functionally active synthetic metalloproteins.     

Self-assemblies of triskelion A2B-type amphiphilic polypeptide showing pH-responsive morphology transformation

A pH-responsive rolled-sheet morphology was prepared from a triskelion A(2)B-type amphiphilic polypeptide having a histidine residue as a pH-responsive unit. The dimensions of the rolled sheet were 85 nm diameter and 210 nm length with a sheet turn number of 2.0 at pH 7.4. Upon decreasing the pH from 7.4 to 5.0, the layer spacing of the rolled sheets was widened from ca. 9 to ca. 19 nm due to electrostatic repulsion caused by histidine protonation. This morphology change occurred reversibly with a pH change between 7.4 and 5.0. The molecular packing in the rolled sheets was shown to be loosened at pH 5.0 on the basis of electron diffraction measurements. The tightness of the rolled sheets was thus controlled reversibly by a pH change due to a single protonation in the amphiphilic polypeptide.     

Biomembrane Model from Stable Polymer Membrane Having Polypeptide Domains as Channels

A novel polymer membrane having a transmembrane permeation pathway (channel) was prepared from a polyvinyl-polypeptide graft copolymer. The transmembrane continuous phases of the hydrophilic polypeptide were found to be formed in the stable matrix from vinyl polymer and to function as a permeation pathway for polar substances; the polypeptide domain is regarded as a membrane protein model. The infrared and circular dichroism spectra of the membrane showed the pH-dependent conformational change of the polypeptide segment. Regulation of permeability and permselectivity was performed by pH based on the conformational transition of the channel-composing polypeptide. In addition, the membrane has been found to respond to divalent cations, cationic surfactants, urea, and organic solvents in terms of membrane permeability as well as conformational status of polypeptide.     

Pulsed Chronopotentiometric Detection of Thrombin Activity Using Reversible Polyion Selective Electrodes

A simple and rapid electrochemical method for the detection of thrombin activity is presented here for the first time using a synthetic polypeptide substrate and a pulsed chronopotentiometry transduction protocol with polyion selective electrodes. A cathodic current applied across a polyion selective membrane electrode causes the extraction of the polypeptides from the sample into the membrane and the membrane potential, which is a function of the concentration of these polypeptides in the sample, is measured at the same time. Since the polyion extraction is a diffusion‐controlled mass transfer process, depletion of the polypeptides at the membrane‐sample interface at a characteristic transition time occurs. The square root of the transition time is directly proportional to the concentration of the polypeptide substrate according to the Sand equation. Upon addition of thrombin, the polypeptide substrate undergoes proteolysis and yields smaller peptide fragments that exhibit smaller measured electromotive force (emf) responses since they are less preferred by the membrane, and the transition times become shorter. The rate of change of the square root of the transition time is directly proportional to the change in the polypeptide concentration, which in turn relates to the polypeptide hydrolysis time, and can be used for monitoring enzyme activity. Indeed, the square root of transition time was found to be linear with the proteolysis time with R²=0.98. The activity of thrombin was determined to be 3.6 μg substrate per μg thrombin per Min.     

Elution mechanism of polypeptides in reversed-phase liquid chromatography based on the critical threshold of organic solvent to induce abrupt change in adsorption capacity to the column packing

Adsorption capacity of polypeptides to the column packing in a solution containing multiple organic solvents was found to be expressed by means of an fn value, which is the sum of the ratios of the content of each organic solvent in the solution to the critical content of each organic solvent to cause abrupt change in the adsorption capacity, and to change abruptly at the point where the fn value becomes 1. Additionally, our results indicate that each polypeptide is eluted by the eluent containing a specific organic solvent content regardless of gradient elution rate in reversed-phase liquid chromatography, and that total organic solvent content in the eluent containing polypeptides is equal to the critical content. Considering the power law relationship between the retention times and the gradient elution rates, our results suggest that the elution of each polypeptide in reversed-phase liquid chromatography is mainly controlled by abrupt change in the adsorption capacity induced by change in the organic solvent content of the eluent during a gradient elution process, and that the abrupt change repeats across the critical threshold while a polypeptide moves through the column, and as a result, each polypeptide is concentrated in the eluent with the critical threshold.     

Spin-label study of the conformational states of polypeptide models of histones

The conformational states of the regular polypeptides (Gly-Lys-Gly)_n, (Ala-Orn-Gly)_n, and (Ala-Orn-Ala)_n have been studied by the spin-label method. Their behavior in solutions of guanidine hydrochloride and urea and in solutions of salts of bivalent metals does not contradict the presence of an extended levohelical conformation in their polypeptide chains. In CaCl₂ (5 N) solutions the polypeptides exhibit aggregation properties. A study of the behavior of the poly peptide at these temperatures has shown that with a rise in temperature there is a monotonic change in the structures of the polypeptide chains that is characteristic for a conformation of the polyproline-II type. Differences have been observed in the behavior of glycine-and alanine-containing polypeptides in the presence of sodium dodecyl sulfate with a change in the temperature.V. I. Nikitin Institute of Chemistry of the Academy of Sciences of the Tadzhik SSR, Dushanbe. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 218–222, March–April, 1986.     

异肽键稳定的MERS-CoV融合蛋白S2亚基N端重复序列三股琢螺旋的构建

通过在天然N肽的氨基端引入可以诱导螺旋三聚体形成的人工多肽序列,并通过酰基转移反应在上述嵌合肽所形成的三股α螺旋间引入异肽键,构建了中东呼吸综合征病毒( MERS-CoV)的N-trimer模型,为MERS-CoV融合抑制剂的设计奠定了基础。     

Multiple polypeptide forms observed in two-dimensional gels of Methylococcus capsulatus (Bath) polypeptides are generated during the separation procedure

We have examined two-dimensional electrophoresis (2-DE) gel maps of polypeptides from the Gram-negative bacterium Methylococcus capsulatus (Bath) and found the same widespread trains of spots as often reported in 2-DE gels of polypeptides of other Gram-negative bacteria. Some of the trains of polypeptides, both from the outer membrane and soluble protein fraction, were shown to be generated during the separation procedure of 2-DE, and not by covalent post-translational modifications. The trains were found to be regenerated when rerunning individual polypeptide spots. The polypeptides analysed giving this type of trains were all found to be classified as stable polypeptides according to the instability index of Guruprasad et al. (Protein Eng. 1990, 4, 155-161). The phenomenon most likely reflects conformational equilibria of polypeptides arising from the experimental conditions used, and is a clear drawback of the standard 2-DE procedure, making the gel picture unnecessarily complex to analyse.     

A whole-cell assay for the high throughput screening of calmodulin antagonists

Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca²⁺, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that are potential antipsychotic drugs.     

ZiCo: a peptide designed to switch folded state upon binding zinc

We describe a novel approach to the design of a metal-triggered conformational switch. Specifically, two distinct protein-folding motifs were merged into one polypeptide sequence. The target structures were an alpha-helical coiled-coil trimer and zinc-bound monomer. Solution-phase spectroscopic, sedimentation, and binding studies confirmed the key aspects of the design. Both forms of the peptide were cooperatively folded, and the switch between them was reversible. This design process potentially presents a novel route to peptide-based biosensors.     

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.