Solution-state NMR Spectroscopy Research Articles

Novel polymeric aggregates of pentaphosphaferrocenes and monocationic coinage metal salts – Synthesis of [{Cp∗Fe(η5-P5)}{Cu(GaCl4)}2]n and [{CpBIGFe(η5-P5)}Ag]n [Al(OC(CF3)3)4

The reaction of pentaphosphaferrocenes [CpRFe(η5-P5)] (CpR=Cp∗=η5-C5Me5, CpBIG=η5-C5(4-nBuC6H4)) with monocationic salts of copper and silver leads to the formation of [{Cp∗Fe(η5-P5)}{Cu(GaCl4)}2]n (2) and [{CpBIGFe(η5-P5)}Ag]n[Al{OC(CF3)3}4]n (3), respectively, representing the rare structural motifs of a 1,2,3,4- or a 1,3-coordination mode of the cyclo-P5 unit. Both the 2D network as well as the 1D polymer are obtained from self-assembly processes and characterized by X-ray structure analysis. Compound 2 exemplifies a sheet-like structure, whose layers are linked by short Cl⋯H-contacts, whereas the structure of 3 is comprised of cationic strands, whose positive charge is balanced by the weakly coordinating anion [Al{OC(CF3)3}4]. Dissociative properties of both polymeric complexes are compared by solution and solid state NMR spectroscopy.

Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour.

α-synuclein (αS) is a protein involved in neurotransmitter release in presynaptic terminals, and whose aberrant aggregation is associated with Parkinson’s disease. In dopaminergic neurons, αS exists in a tightly regulated equilibrium between water-soluble and membrane-associated forms. Here we used a combination of solid-state and solution-state NMR spectroscopy to characterize the conformations of αS bound to lipid membranes mimicking the composition and physical properties of synaptic vesicles. The study evidences three αS regions possessing distinct structural and dynamical properties, including an N-terminal helical segment having a role of membrane-anchor, an unstructured C-terminal region that is weakly associated with the membrane, and a central region acting as a sensor of the lipid properties and determining the affinity of αS membrane binding. Taken together, our data define the nature of the interactions of αS with biological membranes and provide insights into their roles in the function and in the molecular processes leading the aggregation of this protein.

Mapping the binding interactions between a specialized Hsp70 chaperone and its Hsp40 co‐chaperone: a direct role for the Hsp40 HPD motif (567.5)

Proteins from the ISC operon of E. coli constitute the machinery used for the synthesis of iron‐sulfur clusters and their delivery to recipient apo‐proteins. [2Fe‐2S] cluster transfer from the holo‐scaffold protein IscU depends on ATP hydrolysis in the nucleotide‐binding domain (NBD) of HscA, a specialized Hsp70 chaperone. HscB, an Hsp40‐type co‐chaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer. However, the interactions between HscA and HscB have remained uncharacterized, and, moreover, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. We have created three variants of the isolated HscA NBD truncated at different residues within the linker, and have shown that the linker binds to the NBD and autoactivates ATP hydrolysis. Using solution‐state NMR spectroscopy and chemical shift perturbations, we mapped the apo‐HscA, HscA‐ADP and HscA‐ATP binding sites onto HscB. Our NMR data provide the first evidence of a structural role for the ubiquitously conserved J‐domain residues HPD of HscB, which we found selectively bind to the ATP‐bound form of HscA. Additionally, we have identified and characterized a previously unreported interaction between HscA and IscA, an iron‐binding protein putatively involved in [4Fe‐4S] cluster transfer, which competes with HscB for binding to HscA.Grant Funding Source: Supported by NIH grants U01 GM094622 and P41 GM103399

Amphiphilic and Phase‐Separable Ionic Liquids for Biomass Processing

One main limiting factor for the technoeconomics of future bioprocesses that use ionic liquids (ILs) is the recovery of the expensive and potentially toxic IL. We have demonstrated a new series of phase-separable ionic liquids, based on the hydrophobic tetraalkylphosphonium cation ([PRRRR](+)), that can dissolve lignin in the neat state but also hemicellulose and high-purity cellulose in the form of their electrolyte solutions with dipolar aprotic solvents. For example, the IL trioctylmethylphosphonium acetate ([P8881][OAc]) was demonstrated to dissolve up to 19 wt % of microcrystalline cellulose (MCC) at 60 °C with the addition of 40 wt % of DMSO. It was found that the MCC saturation point is dependent on the molar ratio of DMSO and IL in solution. At the optimum saturation, a ∼1:1 molar ratio of [P8881][OAc] to anhydroglucose units is observed, which demonstrates highly efficient solvation. This is attributed to the positive contribution that these more amphiphilic cation-anion pairs provide, in the context of the Lindman hypothesis. This effective dissolution is further illustrated by solution-state HSQC NMR spectroscopy on MCC. Finally, it is also demonstrated that these electrolytes are phase separable by the addition of aqueous solutions. The addition of 10 % NaOAc solution allows a near quantitative recovery of high-purity [P8881][OAc]. However, increased volumes of aqueous solution reduced the recovery. The regenerated material was found to partially convert into the cellulose II crystalline polymorph.

Are Spider Silk Proteins a New Class of Intrinsically Disordered Proteins?

Spider dragline silk is an outstanding biopolymer with a strength that exceeds steel by weight and a toughness greater than high-performance fibers like Kevlar. For this reason, understanding how a spider converts the gel-like, aqueous protein spinning dope within the major ampullate (MA) gland into a super fiber is of great importance for developing future biomaterials based on spider silk. In this work, the initial state of the silk proteins within Black Widow MA glands was probed with solution-state NMR spectroscopy following isotopic enrichment. 15N relaxation parameters, T1, T2 and 15N-{1H} steady-state NOE were measured for twelve backbone environments at two spectrometer frequencies, 500 and 800 MHz. All observed backbone environments are consistent with MA silk protein dynamics on the fast sub-nanosecond timescale. In addition, the observed conformation-dependent isotropic chemical shifts are consistent with a random coil. Therefore, it is concluded that the repetitive core of spider MA proteins are in an unfolded, highly flexible state in the MA gland. We propose that spider silk proteins can be considered intrinsically disordered proteins prior to fiber formation.

Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the κ opioid receptor using a soluble protein scaffold

The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the κ opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble β-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.

Backbone NMR assignments of a topologically knotted protein in urea-denatured state

YbeA is a 3-methylpseudoridine methyltransferase from Escherichia coli that forms a stable homodimer in solution. It is one of the deeply trefoil 31 knotted proteins, of which the knot encompasses the C-terminal helix that threads through a long loop. Recent studies on the knotted protein folding pathways using YbeA have suggested that the protein knot remains present under chemically denaturing conditions. Here, we report (1)H, (13)C and (15)N chemical shift assignments for urea-denatured YbeA, which will serve as the basis for further structural characterisations using solution state NMR spectroscopy with paramagnetic spin labeled and partial alignment media.

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.

Separation of chitosan by degree of acetylation using simple free solution capillary electrophoresis

Chitosan is a biopolymer of increasing significance, as well as a renewable and sustainable material. Its main molecular characteristics are molar mass and degree of acetylation (composition). Precise average degrees of acetylation were measured by quantitative (1)H solution-state NMR spectroscopy. While number-average degrees of acetylation had already been determined by (1)H NMR spectroscopy, weight-average degrees of acetylation are also determined and may be more relevant for some properties, such as mechanical properties. We report the first separation of chitosan according to its degree of acetylation using free solution capillary electrophoresis. Capillary electrophoresis separates chitosan in the 'critical conditions': the molar mass plays little role and the separation is by the degree of acetylation. It characterises the heterogeneity of chitosan samples in terms of composition (dispersity of the distribution of degrees of acetylation). This heterogeneity (broad distribution of degrees of acetylation) cannot be neglected contrary to a common assumption found in the literature. This fast and easy separation will allow establishing a structure-property relationships.

Carbon-dot organic surface modifier analysis by solution-state NMR spectroscopy

Carbon dots (C-dots) represent a new class of carbon-based materials that were discovered recently and have drawn the interest of the scientific community, particularly because of their attractive optical properties and their potential as fluorescent sensors. Investigation of the chemical structure of C-dots is extremely important for correlating the surface modifier composition with C-dot optical properties and allow for structure–properties fine tuning. In this article, we report the structural analysis of the surface modifiers of three different types of C-dot nanoparticles (Cwax, Cws, and Csalt) by use of 1D- and 2D-high-resolution NMR spectroscopy in solution. We unambiguously verify that the structure of the modifier chains remains chemically unchanged during the passivation procedure, and confirm the covalent attachment of the modifiers to the nanoparticle core, which contributes no signal to the solution-state NMR spectra. To our knowledge, this is the first study confirming the full structural assignment of C-dot organic surface modifiers by use of solution NMR spectroscopy.

Solution-State 2D NMR Spectroscopy of Plant Cell Walls Enabled by a Dimethylsulfoxide-d6/1-Ethyl-3-methylimidazolium Acetate Solvent

Lignocellulosic biomass is composed of the polysaccharides cellulose and hemicellulose and the polyphenol lignin. Many current methods for analyzing the structure of lignocelluloses involve a sequential extraction of the material and subsequent analysis of the resulting fractions, which is labor-intensive and time-consuming. The work presented here assesses the dissolution of whole lignocellulosic material, focusing on biomass derived from the perennial bioenergy grass Miscanthus. The solvent dimethylsulfoxide (DMSO)-d6 containing 1-ethyl-3-methylimidazolium acetate ([Emim]OAc) was able to dissolve lignocellulosic material completely and gave high-resolution 2D heteronuclear single quantum coherence (HSQC) NMR spectra of the entire array of wall polymers. Extrapolated time-zero HSQC was applied using DMSO-d6/[Emim]OAc-d14 and enabled quantitative analysis of structural traits of lignocellulose components.

Upregulation of galactose metabolic pathway by N-acetylglucosamine induced endogenous synthesis of galactose in Candida albicans

N-Acetylglucosamine (GlcNAc) is an important signaling molecule that plays multiple roles in Candida albicans. Induction of galactose metabolic pathway by GlcNAc is an intriguing aspect of C. albicans biology. In order to investigate the role of galactose metabolic genes (GAL genes) in presence of GlcNAc, we created knockouts of galactokinase (GAL1) and UDP galactose epimerase (GAL10) genes. These mutants failed to grow on galactose and also showed lower growth rate in presence of GlcNAc. Interestingly, expression of GAL genes in presence of GlcNAc was higher in gal1Δ strain relative to that of wild type strain. Moreover, no GlcNAc induced upregulation of GAL genes was observed in the gal10Δ strain suggesting that UDP galactose epimerase is essential for GlcNAc induced activation of GAL genes. GlcNAc induced expression of GAL genes was also investigated in GlcNAc metabolic pathway triple mutant N216 (hxk1Δ nag1Δ dac1Δ). Interestingly, in this mutant the GAL genes are neither induced nor repressed and remain derepressed as found on a neutral carbon source such as glycerol, suggesting that catabolism of GlcNAc play an important role in the expression of GAL genes. GC/MS analysis of derivatized metabolites revealed a significant accumulation of galactose in the gal1Δ strain while no galactose was detected in gal10Δ and N216 strain. Solution-state NMR spectroscopy using N-acetyl-13C1-glucosamine confirmed the flow of 13C label from GlcNAc to galactose. Thus, internal galactose synthesized via UDP galactose pathway from GlcNAc metabolites acts as the inducer of GAL genes in presence of GlcNAc.

First bisphosphonate hydrogelators: potential composers of biocompatible gels.

Recently, investigation of hydrogels has gained ever increasing attention mostly because of their biomedical and pharmaceutical properties, and novel hydrogelators are constantly studied to find functional applications. Bisphosphonates (BPs) are well-known compounds applicable in different fields but are mostly used in clinics as drugs for bone-related diseases. In this study, a novel class of BP-hydrogelators together with a BP-organogelator was found, and the gelating abilities of the compounds were studied. Several techniques to analyze the structure and the properties of the formed gels were used, including solid state 13C and 31P CPMAS and solution state NMR spectroscopy, IR spectroscopy, PXRD, thermoanalysis, as well as SEM.

Gangliosides Mediate a Two-Step Mechanism of Membrane Disruption by Beta-Amyloid: Initial Pore Formation Followed by Membrane Fragmentation

The mechanism of amyloid-β (Aβ) toxicity has long been a topic of intense debate. One prevailing hypothesis states that cytotoxicity arises through Aβ interacting with and disrupting cell membranes. Here, we demonstrate that membrane disruption by Aβ occurs via a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ(1-40), defects form on the membrane that share many of the properties of the Aβ ion-channel-like pores originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, the fiber-dependent step of membrane disruption by Aβ(1-40) involves a detergent-like mechanism characterized by fragmentation of the membrane that is neither cation ion selective nor blockaded by Zn2+. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. In addition, using a combinational technique of solid-state and solution-state NMR spectroscopy, we show that Aβ(1-40) fibrils are capable of binding to ganglioside-containing membranes, potentially providing a beacon for the mechanism of nonspecific membrane fragmentation. This fiber-dependent membrane fragmentation can only be observed in the presence gangliosides, whereas pore formation by freshly dissolved Aβ(1-40) is weakly observed in their absence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.Sciacca, M. F., Kotler, S. A., Brender, J. R., Chen, J., Lee, D. K., and Ramamoorthy, A. (2012) Biophys J 103, 702-710

Plant cell wall profiling by fast maximum likelihood reconstruction (FMLR) and region-of-interest (ROI) segmentation of solution-state 2D 1H–13C NMR spectra

BackgroundInterest in the detailed lignin and polysaccharide composition of plant cell walls has surged within the past decade partly as a result of biotechnology research aimed at converting biomass to biofuels. High-resolution, solution-state 2D 1H–13C HSQC NMR spectroscopy has proven to be an effective tool for rapid and reproducible fingerprinting of the numerous polysaccharides and lignin components in unfractionated plant cell wall materials, and is therefore a powerful tool for cell wall profiling based on our ability to simultaneously identify and comparatively quantify numerous components within spectra generated in a relatively short time. However, assigning peaks in new spectra, integrating them to provide relative component distributions, and producing color-assigned spectra, are all current bottlenecks to the routine use of such NMR profiling methods.ResultsWe have assembled a high-throughput software platform for plant cell wall profiling that uses spectral deconvolution by Fast Maximum Likelihood Reconstruction (FMLR) to construct a mathematical model of the signals present in a set of related NMR spectra. Combined with a simple region of interest (ROI) table that maps spectral regions to NMR chemical shift assignments of chemical entities, the reconstructions can provide rapid and reproducible fingerprinting of numerous polysaccharide and lignin components in unfractionated cell wall material, including derivation of lignin monomer unit (S:G:H) ratios or the so-called SGH profile. Evidence is presented that ROI-based amplitudes derived from FMLR provide a robust feature set for subsequent multivariate analysis. The utility of this approach is demonstrated on a large transgenic study of Arabidopsis requiring concerted analysis of 91 ROIs (including both assigned and unassigned regions) in the lignin and polysaccharide regions of almost 100 related 2D 1H–13C HSQC spectra.ConclusionsWe show that when a suitable number of replicates are obtained per sample group, the correlated patterns of enriched and depleted cell wall components can be reliably and objectively detected even prior to multivariate analysis. The analysis methodology has been implemented in a publicly-available, cross-platform (Windows/Mac/Linux), web-enabled software application that enables researchers to view and publish detailed annotated spectra in addition to summary reports in simple spreadsheet data formats. The analysis methodology is not limited to studies of plant cell walls but is amenable to any NMR study where ROI segmentation techniques generate meaningful results.Please see Research Article: http://www.biotechnologyforbiofuels.com/content/6/1/46/.

Solution-state NMR investigation of the sorptive fractionation of dissolved organic matter by alkaline mineral soils

Environmental context Dissolved organic matter plays a key role in global carbon cycling and environmental contaminant transport. We use one- and two-dimensional solution-state nuclear magnetic resonance spectroscopy to characterise dissolved organic matter before and after binding to alkaline subsoils with low organic carbon content. The results show that the dissolved organic matter is selectively fractionated through preferential binding of specific organic carbon functional groups. Abstract Sorption to clay minerals is a prominent fate of dissolved organic matter (DOM) in terrestrial environments. Previous studies have observed that DOM is selectively fractionated by interactions with both pure clay minerals and acidic mineral soils. However, the specific DOM functional groups that preferentially sorb to mineral surfaces in alkaline soils require further examination because higher basicity could change the nature of these sorptive interactions. Biosolids-derived DOM was characterised using one- and two-dimensional solution-state NMR spectroscopy before and after sorption to three alkaline subsurface mineral soils with varying mineralogy. Carboxylic DOM components sorbed preferentially to all soils, likely due to cation bridging and ligand exchange mechanisms. Aliphatic constituents were selectively retained only by a soil with high clay mineral content, possibly by van der Waals interactions with montmorillonite surfaces. Polar carbohydrate and peptide components of the DOM did not exhibit preferential sorption and may remain mobile in the soil solution and potentially stimulate microbial activity. A relatively low signal from aromatic DOM components prevented a full assessment of their sorption behaviour. The results suggest that DOM is selectively fractionated by similar interactions in both acidic and alkaline soils that may play a key role in the chemical and biochemical processes of subsurface environments.

Structure and Stability of Duplex DNA Containing (5′S)-5′,8-Cyclo-2′-deoxyadenosine: An Oxidatively Generated Lesion Repaired by NER

Cellular respiration and ionizing radiation generate 5',8-cyclo-2'-deoxyribonucleosides, a special type of DNA damage that involves two modifications in the same nucleotide. These lesions evade the action of base excision glycosylases, and their removal is a function of the nucleotide excision repair pathway. Diastereomeric 5',8-cyclo-2'-deoxyadenosine blocks mammalian DNA replication, diminishes the levels of DNA transcription, and induces transcriptional mutagenesis. Using solution state NMR spectroscopy and restrained molecular dynamics simulations, we have determined the structure of an undecameric DNA duplex having a centrally located (5'S)-5',8-cyclo-2'-deoxyadenosine residue paired to T. The damaged duplex structure is a right-handed helix having Watson-Crick base-pair alignments throughout, and 2-deoxyribose puckers within the B-form conformation. Only small structural perturbations are observed at the lesion-containing and 5'-flanking base pair. The 2-deoxyribose of the damaged nucleotide adopts the O4'-exo conformation, and the S-cdA·T base pair is propeller twisted. The 5'-lesion-flanking base is tilted forming a significantly buckled base pair with its partner guanine. Analysis of UV-melting curves indicates mild thermal and thermodynamic destabilization on the damaged duplex. The S-cdA·T duplex structure shows many similarities to and some intriguing differences from the recently reported structure of an S-cdG·dC duplex³¹ that suggest different lesion site dynamics.

1H, 13C, and 15N backbone resonance assignments of the L124D mutant of StAR-related lipid transfer domain protein 4 (StARD4)

Protein-mediated cholesterol trafficking is central to maintaining cholesterol homeostasis in cells. START (Steroidogenic acute regulatory protein-related lipid transfer) domains constitute a sterol and lipid binding motif and the START domain protein StARD4 typifies a small family of mammalian sterol transport proteins. StARD4 consists of a single START domain and has been reported to act as a general cholesterol transporter in cells. However, the structural basis of cholesterol uptake and transport is not well understood and no cholesterol-bound START domain structures have been reported. We have undertaken the study of cholesterol binding and transport by StARD4 using solution state NMR spectroscopy. To this end, we report nearly complete (1)H, (15)N, and (13)C backbone resonance assignments of an inactive but well behaved mutant (L124D) of StARD4.

Modulation of the neurotensin solution structure in the presence of ganglioside GM1 bicelle

Neurotensin (NT) is an endogenous tridecapeptide neurotransmitter that shows multiple biological function in central and peripheral nervous systems. Gangliosides are glycosphingolipids, most abundant in the plasma membrane of nerve cells. Here we investigate the change of neurotensin solution structure induced by isotropic CHAPS-PC bicelles with and without ganglioside GM1 using solution state NMR spectroscopy. In aqueous solution the peptide is predominately unstructured. In the presence of bicelle overall structure of the peptide is stabilized. In CHAPS-PC bicelle neurotensin adopts 310 helical structure. In the presence of GM1 containing bicelle, the peptide adopts predominately 310 helical structures with small amount of α-helical structure. These results are consistent with the CD spectroscopic results. Neurotensin interacts better with GM1 containing bicelle than that of the CHAPS-PC bicelle. Docking studies between the Neurotensin Receptor3 (NTS3) and different NT conformations also indicated better binding of the NT conformation obtained in presence of GM1-containing bicelles.

Structural insights into the transmembrane domains of human copper transporter 1

The human copper transporter 1 (hCtr1) mediates cellular uptake of copper and Pt-based chemotherapeutic anticancer drugs. In this paper, we determined the three-dimensional structure and oligomerization of the transmembrane domains (TMDs) of hCtr1 in 40% HFIP aqueous solution by using solution-state NMR spectroscopy. We firstly revealed that TMD1 forms an α-helical structure from Gly67 to Glu84 and is dimerized by close packing of its C-terminal helix; TMD2 forms an α-helical structure from Leu134 to Thr155 and is self-associated as a trimer by the hydrophobic contact of TMD2 monomers; TMD3 adopts a discontinuous helix structure, known as 'α-helix-coiled segment-α-helix', and is dimerized by the interaction between the N-terminal helices. The motif GxxxG in TMD3 is not fully involved in the helix, but partially unstructured as a linker between helices. The flexible linker of TMD3 may serve as a gating adapter to mediate pore on and off switch. The differences in the structure and aggregation of the TMD peptides may be related to their different roles in the channel formation and transport function.