Solution NMR Studies Research Articles

Solution NMR studies reveal no global flexibility in the catalytic domain of CDC25B.

The CDC25B phosphatase is a critical regulator of the cell cycle and has been validated as an important therapeutic target in cancer. Previous studies using molecular dynamics simulations have concluded that the catalytic domain of CDC25B may experience a significant degree of dynamics or be partially disordered in solution, a finding that has a pronounced impact on the structure-based development of CDC25B inhibitors. We have probed the backbone dynamics of the CDC25B catalytic domain in solution using NMR relaxation experiments and found that the core of the protein is relatively rigid and does not experience any large-scale dynamics over a broad range of time scales. Furthermore, based on residual dipolar coupling measurements we have concluded that the conformation in solution is very similar to that observed in the crystal form. Importantly, these findings rationalize the application of the CDC25B crystal structure in structure-based drug development.

Solution and Solid-State Structure of cis-Dichloro-(N,N′-dimethylethylenediamine)platinum(II)

The chiral coordination compound cis-dichloro-(N,N′-dimethylethylenediamine) platinum(II) (Pt(dmen)Cl2) has been synthesized and characterized by NMR and X-ray crystallography. NMR studies in d 6 -DMSO solution reveal distinct resonances are observed for the equatorial and axial methylene protons leading to the conclusion that Pt(dmen)Cl2 is found as one stereoisomer and one conformer of the five-membered ring formed by the Pt2+ and dmen ligand. In D2O NMR evidence also suggests one stereoisomer and one conformer dominates in aqueous solution. Crystallographic studies indicate the presence of the R,R′/S,S′ stereoisomer with a λ conformation of the Pt2+-dmen five-membered ring. This implies that the stereoisomer and conformer observed in d 6 -DMSO and D2O solution are likely the same. The crystal structure of Pt(dmen)Cl2 has the unusual feature of homochiral one-dimensional stacks of R,R′-Pt(dmen)Cl2 and S,S′-Pt(dmen)Cl2. Structure of Pt(dmen)Cl2: monoclinic, P 21/c, a = 7.491(2) A, b = 16.356(4) A, c = 14.503(3) A, β = 103.636(8)°, V = 1726.8(7) A3, Z = 8, GOF = 1.121, R 1 = 0.0514, wR 2 = 0.1298. cis-Dichloro-(N,N′-dimethylethylenediamine)platinum(II) forms a racemic mixture of the S,S′ and R,R′ enantiomers in solution as well as in the solid state where the crystalline packing is characterized by homochiral stacks.

Capping motifs stabilize the leucine‐rich repeat protein PP32 and rigidify adjacent repeats

Capping motifs are found to flank most β-strand-containing repeat proteins. To better understand the roles of these capping motifs in organizing structure and stability, we carried out folding and solution NMR studies on the leucine-rich repeat (LRR) domain of PP32, which is composed of five tandem LRR, capped by α-helical and β-hairpin motifs on the N- and C-termini. We were able to purify PP32 constructs lacking either cap and containing destabilizing substitutions. Removing the C-cap results in complete unfolding of PP32. Removing the N-cap has a much less severe effect, decreasing stability but retaining much of its secondary structure. In contrast, the dynamics and tertiary structure of the first two repeats are significantly perturbed, based on (1)H-(15)N relaxation studies, chemical shift perturbations, and residual dipolar couplings. However, more distal repeats (3 to C-cap) retain their native tertiary structure. In this regard, the N-cap drives the folding of adjacent repeats from what appears to be a molten-globule-like state. This interpretation is supported by extensive analysis using core packing substitutions in the full-length and N-cap-truncated PP32. This work highlights the importance of caps to the stability and structural integrity of β-strand-containing LRR proteins, and emphasizes the different contributions of the N- and C-terminal caps.

Encapsulation of a metal complex within a self-assembled nanocage: synergy effects, molecular structures, and density functional theory calculations.

A novel palladium-based metallacage was self-assembled. This nanocage displayed two complementary effects that operate in synergy for guest encapsulation. Indeed, a metal complex, [Pt(NO2)4](2-), was hosted inside the cavity, as demonstrated by solution NMR studies. Single-crystal X-ray diffraction shows that the guest adopts two different orientations, depending on the nature of the host-guest interactions involved. A density functional theory computational study is included to rationalize this type of host-guest interaction. These studies pave the way to a better comprehension of chemical interaction and transformation within confined nanospaces.

Experimental and Theoretical Studies on Arene‐Bridged Metal–Metal‐Bonded Dimolybdenum Complexes

The bis(hydride) dimolybdenum complex, [Mo2(H)2{HC(N-2,6-iPr2C6H3)2}2(thf)2], 2, which possesses a quadruply bonded Mo2(II) core, undergoes light-induced (365 nm) reductive elimination of H2 and arene coordination in benzene and toluene solutions, with formation of the Mo(I)2 complexes [Mo2{HC(N-2,6-iPr2C6H3)2}2(arene)], 3⋅C6H6 and 3⋅C6H5Me, respectively. The analogous C6H5OMe, p-C6H4Me2, C6H5F, and p-C6H4F2 derivatives have also been prepared by thermal or photochemical methods, which nevertheless employ different Mo2 complex precursors. X-ray crystallography and solution NMR studies demonstrate that the molecule of the arene bridges the molybdenum atoms of the Mo(I)2 core, coordinating to each in an η(2) fashion. In solution, the arene rotates fast on the NMR timescale around the Mo2-arene axis. For the substituted aromatic hydrocarbons, the NMR data are consistent with the existence of a major rotamer in which the metal atoms are coordinated to the more electron-rich C-C bonds.

Effect of trisubstituted imidazolium based ionic liquid 1-butyl-2,3-dimethylimidazolium chloride on the aggregation behaviour of sodium dodecylsulphate in aqueous media

Effect of 1-butyl-2,3-dimethylimidazolium chloride [bdmim][Cl], a trisubstituted ionic liquid on the critical micelle concentration (cmc) of sodium dodecylsulphate (SDS) in aqueous medium have been studied using conductometry from 298.15 to 318.15K and pyrene fluorescence probe analysis at 298.15K. Temperature and ionic liquid concentration dependent variation in cmc been discussed pertaining to the changes in degree of dissociation (α) and thermodynamic parameters viz. standard free energy (ΔGm°) and entropy (ΔSm°) of micellization. NMR studies of post-micellar solutions have been carried out to understand the various intermolecular interactions present in these systems.

Influence of Fluorination and Boronic Group Synergy on the Acidity and Structural Behavior of o-Phenylenediboronic Acids

The solid-state and solution structural properties and acidity of a series of fluorinated 1,2-phenylenediboronic acids were investigated. Solution NMR studies indicate that these compounds equilibrate with their dehydrated forms, in the simplest case presumably possessing the cyclic benzoxadiborole structure. Ab initio calculations showed that the stability of such cyclic semianhydrides is improved by fluorination of the aromatic ring and complexation of one of the boron centers with water. This was demonstrated by the crystal structure determination of tetrafluoro-1,2-phenylenediboronic acid. The coordinated water molecule participates in very strong intermolecular hydrogen bonding with the OH group bonded to the four-coordinate boron center (dO···O = 2.423(2) A, Eint = −87 kJ mol–1). This indicates that in fact this compound is an oxonium, i.e., Bronsted acid, which is exceptional for boronic acids. Under different crystallization conditions, tetrafluoro-1,2-phenylenediboronic acid dimerizes by aggregat...

Ru(II) trithiacyclononane 5-(2-hydroxyphenyl)-3-[(4-methoxystyryl)pyrazole], a complex with facile synthesis and high cytotoxicity against PC-3 and MDA-MB-231 cells

The ruthenium(II) complex [Ru([9]aneS 3)(phpz)Cl 2] (1) ([9]aneS 3=trithiacyclononane, phpz=5-(2- hydroxyphenyl)-3-[(4-methoxystyryl)pyrazole]) was readily isolated by reacting [Ru([9]aneS 3)(DMSO)Cl 2] with one equivalent of the ligand phpz. A combination of MS, FT–IR and solution NMR studies (1-D and 2-D) was employed to determine the structural formula of the complex 1, in which phpz coordinates in a monodentate mode to Ru(II) by a simple replacement of the leaving group DMSO of the precursor. The cytotoxic properties of 1 in vitro were investigated by determination of the half-maximal growth inhibition on the human prostate (PC-3) and breast cancer cells (MDA-MB-231).

Optimized Reverse Micelle Surfactant System for High-Resolution NMR Spectroscopy of Encapsulated Proteins and Nucleic Acids Dissolved in Low Viscosity Fluids

An optimized reverse micelle surfactant system has been developed for solution nuclear magnetic resonance studies of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. Comprising the nonionic 1-decanoyl-rac-glycerol and the zwitterionic lauryldimethylamine-N-oxide (10MAG/LDAO), this mixture is shown to efficiently encapsulate a diverse set of proteins and nucleic acids. Chemical shift analyses of these systems show that high structural fidelity is achieved upon encapsulation. The 10MAG/LDAO surfactant system reduces the molecular reorientation time for encapsulated macromolecules larger than ∼20 kDa leading to improved overall NMR performance. The 10MAG/LDAO system can also be used for solution NMR studies of lipid-modified proteins. New and efficient strategies for optimization of encapsulation conditions are described. 10MAG/LDAO performs well in both the low viscosity pentane and ultralow viscosity liquid ethane and therefore will serve as a general surfactant system for initiating solution NMR studies of proteins and nucleic acids.

Binding mode of the activity‐modulating C‐terminal segment of NS2B to NS3 in the dengue virus NS2B–NS3 protease

The two-component dengue virus NS2B-NS3 protease (NS2B-NS3pro) is an established drug target but inhibitor design is hampered by uncertainties about its 3D structure in solution. Crystal structures reported very different conformations for the functionally important C-terminal segment of the NS2B cofactor (NS2Bc), indicating open and closed conformations in the absence and presence of inhibitors, respectively. An earlier NMR study in solution indicated that a closed state is the preferred conformation in the absence of an artificial linker engineered between NS2B and NS3pro. To obtain direct structural information on the fold of unlinked NS2B-NS3pro in solution, we tagged NS3pro with paramagnetic tags and measured pseudocontact shifts by NMR to position NS2Bc relative to NS3pro. NS2Bc was found to bind to NS3pro in the same way as reported in a previously published model and crystal structure of the closed state. The structure is destabilized, however, by high ionic strength and basic pH, showing the importance of electrostatic forces to tie NS2Bc to NS3pro. Narrow NMR signals previously thought to represent the open state are associated with protein degradation. In conclusion, the closed conformation of the NS2B-NS3 protease is the best model for structure-guided drug design.

Ring‐opening polymerization of cyclic esters by pincer complexes derived from alkaline earth metals

We report the synthesis and characterization of new Ca(II) and Mg(II) complexes of the type [Ph―PPP]MR (M = Ca, R = N(SiMe3)2 (1); M = Mg, R = nBu (2)) where Ph―PPP− is a tridentate monoanionic ligand (Ph―PPP―H = bis(2‐diphenylphosphinophenyl)phosphine). Reaction of the opportune metal precursor (Ca[N(SiMe3)2]2.THF2 or MgnBu2) with 1 equiv. of the pro‐ligand Ph―PPP―H produces the corresponding calcium amido (1) or magnesium butyl (2) complex in high yield. Solution NMR studies show monomeric and kinetically stable structures for both species. The obtained complexes efficiently mediate the ring‐opening polymerization of ɛ‐caprolactone showing a turnover frequency of 40 000 h−1. In the presence of a hexogen alcohol, up to 2000 equiv. of monomer are converted by using a low loading of catalyst (5 µmol). Kinetic studies show a first‐order reaction in monomer concentration. Copyright © 2014 John Wiley & Sons, Ltd.

A Ditopic Catechol Phosphane as Building Block for Selective Construction of Heterometallic Complexes with Early and Late Transition Metal Centers

Base-assisted reaction of catechol phosphane 2 (H2L) with [M′Cl2(cod)] (cod = 1, 5-cyclooctadiene, M′ = Pd, Pt) yielded chelate complexes [M′(HL)2] (7a, b). Spectroscopic and single-crystal X-ray diffraction studies revealed that both complexes feature cis-configuration of the P- and O-donor atoms in solution and in the solid state. Reaction of 7a, b with acetylacetonato or alkoxide complexes [MO2(acac)2] (M = Mo, W), [VO(acac)2], [{Ti(μ-O)(acac)2}2], or Ti(OiPr)4 gave good to excellent yields of early-late heterometallic complexes [MOn(μ-L)2M′] (MOn = MoO2, WO2, VO; 8a, b–10a, b) or [Ti(RO-1κO)2(μ-L-1κ2O, O'-2κ2P, O)2Pd] (R = Me, iPr; 11a, b), which were inaccessible via other synthetic routes. Spectroscopic and single-crystal X-ray diffraction studies revealed that the early metal centres in 8a, b, 9b and in 11b feature distorted octahedral coordination spheres with rigid transoid alignment of the catechol ring planes. Vanadium complexes 10a, b exhibit a square-pyramidal coordination sphere with cisoid alignment of the catechol ring planes and evidence for intermolecular pairing via weak VO···Pd contacts in the solid state; complexes 8, 9 do not undergo conformational inversion on the NMR time-scale. The molecular structure of Ti complex 11a is characterized by a different orientation of the catechol moieties, which can be envisaged to picture an intermediate state during a configuration inversion process, and a strong hydrogen bridge between a terminally coordinated catecholato-oxygen atom and a solvent molecule (MeOH). Solution NMR studies indicate that the (MeO)2Ti(μ-L)2M' framework is in this case conformationally labile and that the MeO ligands undergo intermolecular dynamic exchange with the solvent.

Nature of Si–H Interactions in a Series of Ruthenium Silazane Complexes Using Multinuclear Solid-State NMR and Neutron Diffraction

Three new N-heterocyclic-silazane compounds, 1a-c, were prepared and employed as bidentate ligands to ruthenium, resulting in a series of [Ru(H){(κ-Si,N-(SiMe2-N-heterocycle)}3] complexes (3a-c) featuring the same RuSi3H motif. Detailed structural characterization of the RuSi3H complexes with X-ray diffraction, and in the case of triazabicyclo complex [Ru(H){κ-Si,N-(SiMe2)(C7H12N3)}3] (3a), neutron diffraction, enabled a reliable description of the molecular geometry. The hydride ligand of (3a) is located closer to two of the silicon atoms than it is to the third. Such a geometry differs from that of the previously reported complex [Ru(H){(κ-Si,N-(SiMe2)N(SiMe2H)(C5H4N)}3] (3d), also characterized by neutron diffraction, where the hydride was found to be equidistant from all three silicon atoms. A DFT study revealed that the symmetric and less regular isomers are essentially degenerate. Information on the dynamics and on the Ru···H···Si interactions was gained from multinuclear solid-state ((1)H wPMLG, (29)Si CP MAS, and 2D (1)H-(29)Si dipolar HETCOR experiments) and solution NMR studies. The corresponding intermediate complexes, [Ru{κ-Si,N-(SiMe2-N-heterocycle)}(η(4)-C8H12)(η(3)-C8H11)] (2a-c), involving a single silazane ligand were isolated and characterized by multinuclear NMR and X-ray diffraction. Protonation of the RuSi3H complexes was also studied. Reaction of 3a with NH4PF6 gave rise to [Ru(H)(η(2)-H -SiMe2)κ-N-(C7H12N3){κ-Si,N-(SiMe2)(C7H12N3)}2](+)[PF6](-)(4aPF6) which was isolated and characterized by NMR spectroscopy, X-ray crystallography, and DFT studies. The nature of the Si-H interactions in this silazane series was analyzed in detail.

Preparation of Pt-Tl clusters showing new geometries. X-ray, NMR and luminescence studies.

Square planar complexes [Pt(CNC)L] (CNC = C,N,C-2,6-NC5H3(C6H4-2)2; L = tht (tetrahydrothiophene, SC4H8, 1), L = CN(t)Bu (2)) react with TlPF6 in different Pt/Tl molar ratios (3/1 in the case of 1 and 1/1 in the case of 2) yielding the complexes [{Pt(CNC)(tht)}3Tl](PF6) (3) and [Pt(CNC)(CN(t)Bu)Tl](PF6) (4), respectively. The structures of 3 and 4 (X-ray) show the presence of Pt→Tl dative bonds unsupported by any bridging ligands. In complex 3, the only Tl centre is simultaneously bonded to three Pt atoms forming a perfect equilateral triangle with Pt-Tl distances of 2.9088(5) Å, remarkably short. Complex 4 is formed by three "Pt(CNC)(CN(t)Bu)Tl" units, disposed in a triangular fashion, linked together through η(6)-Tl-arene interactions, and showing Pt-Tl bonds with distances of ca. 3.04 Å. The study of these crystal structures would seem to indicate that the difference between the Pt/Tl ratios found in the complexes 3 and 4 is due to the steric requirements of the L ligand bonded to Pt. NMR studies both in solution and in the solid state show that the Pt-Tl bond persists in solutions of 3 and 4. The UV-vis spectra of 3 and 4 in solution display the same profiles as those of 1 and 2, which may suggest a partial dissociation of the Pt-Tl bond in solution. However, by DFT calculations it was proved that in this case the formation of the Pt-Tl dative bond does not produce the expected blue-shift in the UV-vis absorptions. The emissive behaviour of 1-4 in the solid state and in frozen solutions is also studied and included in this work.

Ethyl 2-hydroxy-2-methylpropanoate derivatives of magnesium and zinc. The effect of chelation on the homo- and copolymerization of lactide and ε-caprolactone

The preparation, single crystal and molecular structures of the compounds (BDI)Mg(OCMe2COOEt)(L) are described where BDI = 2-[(2,6-diisopropylphenyl)amino]-4-[(2,6-diisopropylphenyl)imino]pent-2-ene and L = pyridine (py) and 4-dimethylaminopyridine (DMAP). In the solid-state the molecules have five coordinate Mg(2+) ions that may be considered as derived from a trigonal bipyramid where the chelating BDI and OCMe2COOEt ligands span equatorial-axial sites. IR spectroscopic data obtained in CH2Cl2 indicate that the chelating nature of the OCMe2COOEt ligand is maintained in solution and variable temperature NMR studies reveal that the ligands py or DMAP undergo a rapid reversible dissociation in toluene-d8 and especially in THF-d8. This leads to a facile exchange involving coordinated and free L upon the addition of L and evidence is presented to support the view that this occurs exclusively by a dissociative interchange mechanism. Zinc forms a related complex (BDI)Zn(OCMe2COOEt) lacking additional coordination of L but maintaining the chelating nature of the OCMe2COOEt ligand and undergoing reversible coordination of L in the presence of added ligands L. The magnesium and zinc complexes will initiate and sustain the living polymerization of lactide (L- or rac-LA) but not the ring-opening polymerization of ε-caprolactone.

Modular syntheses of H4octapa and H2dedpa, and yttrium coordination chemistry relevant to86Y/90Y radiopharmaceuticals

The ligands H2dedpa, H4octapa, p-SCN-Bn-H2dedpa, and p-SCN-Bn-H4octapa were synthesized using a new protection chemistry approach, with labile tert-butyl esters replacing the previously used methyl esters as protecting groups for picolinic acid moieties. Additionally, the ligands H2dedpa and p-SCN-Bn-H2dedpa were synthesized using nosyl protection chemistry for the first time. The use of tert-butyl esters allows for deprotection at room temperature in trifluoroacetic acid (TFA), which compares favorably to the harsh conditions of refluxing HCl (6 M) or LiOH that were previously required for methyl ester cleavage. H4octapa has recently been shown to be a very promising (111)In and (177)Lu ligand for radiopharmaceutical applications; therefore, coordination chemistry studies with Y(3+) are described to assess its potential for use with (86)Y/(90)Y. The solution chemistry of H4octapa with Y(3+) is shown to be suitable via solution NMR studies of the [Y(octapa)](-) complex and density functional theory (DFT) calculations of the predicted structure, suggesting properties similar to those of the analogous In(3+) and Lu(3+) complexes. The molecular electrostatic potential (MEP) was mapped onto the molecular surface of the DFT-calculated coordination structures, suggesting very similar and even charge distributions between both the Lu(3+) and Y(3+) complexes of octapa(4-), and coordinate structures between 8 (ligand only) and 9 (ligand and one H2O). Potentiometric titrations determined H4octapa to have a formation constant (log K(ML)) with Y(3+) of 18.3 ± 0.1, revealing high thermodynamic stability. This preliminary work suggests that H4octapa may be a competent ligand for future (86)Y/(90)Y radiopharmaceutical applications.

Structure Refinement of the Transmembrane Domain (TMD) of KCNE1 Protein using Deer Spectroscopy

KCNE1 is a single trans-membrane protein of the KCNE family that modulates the function of voltage gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden death syndrome, and deafness. However, the structure of its physiologically relevant more native membrane bound state is unknown. The transmembrane domain of KCNE1 plays a key role in mediating the physical association with KCNQ1, modulating KCNQ1 gating kinetics, and modulating KCNQ1 pore conductance. This mechanism is still not fully understood. Previous solution NMR study indicated that KCNE1 possesses a curvilinear transmembrane domain in LMPG micelles. In this study, we used Double electron electron resonance spectroscopy coupled with site-directed spin labeling and simulated annealing molecular dynamics to report the structure of the TMD of KCNE1 in proteoliposomes. High quality DEER data consisting of similar distances in micelles, proteoliposomes and lipodisq nanoparticles suggest a curved helical TMD of KCNE1 in proteoliposomes similar to that in micelles. Additionally, we employed DEER distance restraints obtained at 8 double spin labeling sites in simulated annealing molecular dynamics and predicted the structure of the TMD of KCNE1 in vesicles which supports the previous micelle structure. This study will provide useful information about how TMD functions in channel activation during E1/Q1 interactions in diseases causing mutations.

The Molecular Chaperone Hsp70 Activates Protein Phosphatase 5 (PP5) by Binding the Tetratricopeptide Repeat (TPR) Domain

Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development.

Modelling the Interactions of Equinatoxin II with Micelles

Equinatoxin II (EqtII) is a 179-residue toxin from the venom of the sea anemone Actinia equina. EqtII is a member of the actinoporin family of proteins, which have potent lytic activity towards membranes containing sphingomyelin (SM). To gain insight into the atomic-level details governing SM selectivity, a series of all-atom molecular dynamics simulations were performed to model the binding of EqtII to micelles of n-dodecylphosphocholine (DPC) and DPC/SM. These models are in good agreement with concurrent high-resolution solution NMR studies and prior data [1, 2] that suggests membrane binding is dependent on a conserved cluster of aromatic amino acids. From this groundwork study, further simulations will be performed to investigate EqtII oligomerisation and membrane insertion to determine the mechanism of pore formation.

Modelling the Interactions of Equinatoxin II with Micelles

Equinatoxin II (EqtII) is a 179-residue toxin from the venom of the sea anemone Actinia equina. EqtII is a member of the actinoporin family of proteins, which have potent lytic activity towards membranes containing sphingomyelin (SM). To gain insight into the atomic-level details governing SM selectivity, a series of all-atom molecular dynamics simulations were performed to model the binding of EqtII to micelles of n-dodecylphosphocholine (DPC) and DPC/SM. These models are in good agreement with concurrent high-resolution solution NMR studies and prior data [1, 2] that suggests membrane binding is dependent on a conserved cluster of aromatic amino acids. From this groundwork study, further simulations will be performed to investigate EqtII oligomerisation and membrane insertion to determine the mechanism of pore formation.