Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic resonance (NMR) and polarized attenuated total internal reflectance Fourier transform infrared (PATIR-FTIR) properties of melittin are calculated from the trajectory to characterize the orientation of the peptide relative to the bilayer. The residue Lys7 located in the hydrophobic moiety of the helix and residues Lys23, Arg24, Gln25, and Gln26 at the C-terminus hydrophilic form hydrogen bonds with water molecules and with the ester carbonyl groups of the lipids, suggesting their important contribution to the stability of the helix in the bilayer. Lipid acyl chains are closely packed around melittin, contributing to the stable association with the membrane. Calculated density profiles and order parameters of the lipid acyl chains averaged over the molecular dynamics trajectory indicate that melittin has effects on both layers of the membrane. The presence of melittin in the upper layer causes a local thinning of the bilayer that favors the penetration of water through the lower layer. The energetic factors involved in the association of melittin at the membrane surface are characterized using an implicit mean-field model in which the membrane and the surrounding solvent are represented as structureless continuum dielectric material. The results obtained by solving the Poisson-Bolztmann equation numerically are in qualitative agreement with the detailed dynamics. The influence of the protonation state of the N-terminus of melittin is examined. After 600 ps, the N-terminus of melittin is protonated and the trajectory is continued for 400 ps, which leads to an important penetration of water molecules into the bilayer. These observations provide insights into how melittin interacts with membranes and the mechanism by which it enhances their lysis.     

Molecular modeling of vimentin filament assembly

Intermediate-filament forming proteins are known to form rod-shaped dimers that are calculated to be 45 nm in length. Molecular modeling indicates that the dimerization is promoted by interchain hydrophobic interactions between sections of α helix β and helix. Further aggregation involves the formation of tetramers in which two dimers are anti-parallel and staggered to two characteristic degrees of overlap. Modeling indicated that the degrees of stagger are dictated by the association of sections of α helix in 4-chain bundles, in which hydrophobic side chains are sequestered from contact with water. The staggered arrangement of two dimers produces a tetramer having sections of 2-chain rod in which hydrophobic side chains are exposed to water. Extension of the tetramer to form protofilaments may be driven by associations with the 2-chain regions that reduce aqueous exposure of the hydrophobic side chains. Exposure of hydrophobic groups may be reduced by the 2-chain regions folding back upon themselves so that the entire tetramer becomes a 4-chain conformation. This prediction is in line with electron microscope data showing that mixtures of the lower oligomers contain rods of uniform thickness ranging upwards from 45 nm in a series having incremental increases in length. Data from previous chemical crosslinking studies support this model and also the idea that the completed intermediate filaments each consist of seven 4-chain protofilaments. Proteins 26:472–478 © 1996 Wiley-Liss, Inc.     

The structure of melittin in membranes.

The conformation of the polypeptide melittin in lipid membranes as determined by Raman spectroscopy is a bent alpha-helix formed by the mainly hydrophobic residues 1-21, and a nonhelical COOH-terminal segment of the hydrophilic residues 22-26. Fluorescence quenching experiments on residue Trp19 reveal that all COOH-termini are located on that side of a vesicular membrane to which melittin was added. By means of fluorescence energy transfer between unmodified and modified Trp19 residues, melittin is shown to aggregate in membranes predominantly in the form of tetramers. These and previous results on the location and orientation of melittin permit the development of a model for the structure of melittin tetramers in membranes. The hydrophilic sides of four bilayer-spanning helices face each other to form a hydrophilic pore through the membrane.     

Coarse-grained molecular dynamics of tetrameric transmembrane peptide bundles within a lipid bilayer

The conformations of model transmembrane peptides are studied to understand the structural and dynamical aspects of tetrameric bundles using a series of coarse grain (CG) molecular dynamics (MD) simulations since membrane proteins play a crucial role in cell function. In this work, two different amphipathic models have been constructed using similar hydrophobic/hydrophilic characteristics with two structurally distinct morphologies to evaluate the effect of roughness and hydrophilic topology on the structure of tetrameric bundles, one class that forms an ion-channel and one class that does not. Free energy calculations of typical amphipathic peptide topologies show that using a relatively smooth surface morphology allows for a stable conformation of the tetramer bundle in a diamond formation. However, the model with side chains attached to the core in order to roughen the surface has a stable square tetramer bundle which is consistent with experimental data and all-atom (AA) MD simulations. Comparisons of the CG simulations with AA MD simulations are in reasonable agreement with the formation of tetrameric homo-oligomers, partitioning within the lipid bilayer and tilt angle with respect to the bilayer normal. We concluded that a square or diamond shape tetrameric homo-oligomers could be stabilized by rational design of the peptide morphology and topology of the surface, thus allowing us to tune the permeability of the bundle or channel.     

Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer

The crystal structure of a polypeptide chain fragment from the surface layer protein tetrabrachion from Staphylothermus marinus has been determined at 1.8 A resolution. As proposed on the basis of the presence of 11-residue repeats, the polypeptide chain fragment forms a parallel right-handed coiled coil structure. Complementary hydrophobic interactions and complex networks of surface salt bridges result in an extremely thermostable tetrameric structure with remarkable properties. In marked contrast to left-handed coiled coil tetramers, the right-handed coiled coil reveals large hydrophobic cavities that are filled with water molecules. As a consequence, the packing of the hydrophobic core differs markedly from that of a right-handed parallel coiled coil tetramer that was designed on the basis of left-handed coiled coil structures.     

Melittin and the 8-26 fragment. Differences in ionophoric properties as measured by monolayer method

Melittin is a major (approximately 50%) protein component of bee venom. This peptide is an amphiphilic protein, because, while the amino acid residues 1-20 are predominantly hydrophobic (with the exception of Lys-7), residues 21-26 are hydrophilic. The binding properties to vesicles and lipid bilayers of melittin have provided much useful information regarding biological (hemolytic) activity (Habermann, E., 1972, Science [Wash. DC], 177:314-322). Recent studies have convincingly established that the melittin monolayer (at air-water interface) model membrane system allows one to analyze the various forces present in such structures. We present comparative monolayer studies of melittin and the peptide fragment 8-26 regarding the channel formation for the selective anion (Cl-) penetration in monolayers, analogous to melittin (tetramer) channel function in lipid bilayer. The differences in surface pressure and surface potential of monolayers between native melittin and the 8-26 fragment suggest that these may be ascribed to Lys-7.     

The conformation of the pore region of the M2 proton channel depends on lipid bilayer environment

The M2 protein from influenza A virus is a 97-amino-acid protein with a single transmembrane helix that forms proton-selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix tilt, with proposed tilts ranging from approximately 15 degrees to 38 degrees . An important issue for understanding the structure of M2TM is the role of peptide-lipid interactions in the stabilization of the lipid bilayer bound tetramer. Here, we labeled the N terminus of M2TM with a nitroxide and studied the tetramer reconstituted into lipid bilayers of different thicknesses using EPR spectroscopy. Analyses of spectral changes provide evidence that the lipid bilayer does influence the conformation. The structural plasticity displayed by M2TM in response to membrane composition may be indicative of functional requirements for conformational change. The various structural models for M2TM proposed to date--each defined by a different set of criteria and in a different environment--might provide snapshots of the distinct conformational states sampled by the protein.     

Free-energy determinants of alpha-helix insertion into lipid bilayers.

A detailed treatment is provided of the various free-energy terms that contribute to the transfer of a polyalanine alpha-helix from the aqueous phase into lipid bilayers. In agreement with previous work, the hydrophobic effect is found to provide the major driving force for helix insertion. However, an opposing effect of comparable magnitude is also identified and is attributed to the large free-energy penalty associated with the desolvation of peptide hydrogen bonds on transfer to the low dielectric environment of the bilayer. Lipid perturbation effects as well as the entropy loss associated with helix immobilization in the bilayer are also evaluated. Two configurations of a membrane-bound 25mer polyalanine helix were found to be lower in free energy than the isolated helix in the aqueous phase. The first corresponds to the case of vertical insertion, in which a helix terminus protrudes from each side of the bilayer. The second minimum is for the case of horizontal insertion, for which the helix is adsorbed upon the surface of the bilayer. The calculated free-energy minima are found to be in good agreement with recent measurements of related systems. Large free-energy barriers resulting from desolvation of unsatisfied hydrogen-bonding groups at the helix termini are obtained for both insertion processes. The barriers for insertion are significantly reduced if the helix termini are assumed to be "capped" through the formation of hydrogen bonds with polar sidechains. For uncapped helices, our results support recently proposed models in which helices are inserted by first adsorbing on the membrane surface and then having one terminus "swing around" so as to penetrate the bilayer.     

Characterization of selectively 13C-labeled synthetic melittin and melittin analogues in isotropic solvents by circular dichroism, fluorescence, and NMR spectroscopy

The spectroscopic and functional characterization of 13C-labeled synthetic melittin and three analogues is described. Selectively 13C-enriched tryptophan ( [13C delta 1]-L-Trp) and glycine ( [13C alpha]Gly) were incorporated into melittin and three analogues by de novo peptide synthesis. 13C-Labeled tryptophan was incorporated into melittin at position 19 and into single-tryptophan analogues of melittin at positions 17, 11, and 9, respectively. Each of the synthetic peptides contained 13C-labeled glycine at position 12 only. The peptides were characterized functionally in a cytolytic assay, and spectroscopically by CD, fluorescence, and NMR. The behavior of 13C-labeled synthetic melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. All of the analogues were found to be efficient lytic agents and thus were functionally similar to the native peptide, yet no evidence was found for formation of a melittin-like tetramer by any of the analogues in aqueous media, although there was a propensity for apparently nonspecific peptide aggregation, especially for MLT-W9. Since the analogues did exhibit fractional helicities by CD comparable to or even greater than melittin itself in the presence of methanol, we infer that tetramer assembly requires not only the ability to form alpha-helix but also a very precise packing of amino acid side chains of the constituent monomers. The 13C chemical shift of the Gly-12 C alpha was found to be a sensitive marker for helix formation in all of the peptides. For melittin itself, 13C NMR spectra revealed a downfield shift of approximately 1.8 ppm for the Gly-12 13C alpha resonance of the tetramer relative to that observed for the free monomer in D2O. In mixed samples containing melittin monomer and tetramer, two discrete Gly-12 13C alpha peaks were observed simultaneously, suggestive of slow exchange between the two species. We conclude that melittin's ability to form a soluble tetramer is not a prerequisite for cytolytic activity, nor is cytolytic potential precisely correlated with the ability to form an amphiphilic helix.     

Interaction of the Mechanosensitive Channel,MscS, with the Membrane Bilayer through Lipid Intercalation into Grooves and Pockets

All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein–lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even “conservative” hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4.     

Surface binding of alamethicin stabilizes its helical structure: molecular dynamics simulations.

Alamethicin is an amphipathic alpha-helical peptide that forms ion channels. An early event in channel formation is believed to be the binding of alamethicin to the surface of a lipid bilayer. Molecular dynamics simulations are used to compare the structural and dynamic properties of alamethicin in water and alamethicin bound to the surface of a phosphatidylcholine bilayer. The bilayer surface simulation corresponded to a loosely bound alamethicin molecule that interacted with lipid headgroups but did not penetrate the hydrophobic core of the bilayer. Both simulations started with the peptide molecule in an alpha-helical conformation and lasted 2 ns. In water, the helix started to unfold after approximately 300 ps and by the end of the simulation only the N-terminal region of the peptide remained alpha-helical and the molecule had collapsed into a more compact form. At the surface of the bilayer, loss of helicity was restricted to the C-terminal third of the molecule and the rod-shaped structure of the peptide was retained. In the surface simulation about 10% of the peptide/water H-bonds were replaced by peptide/lipid H-bonds. These simulations suggest that some degree of stabilization of an amphipathic alpha-helix occurs at a bilayer surface even without interactions between hydrophobic side chains and the acyl chain core of the bilayer.     

Molecular dynamics simulations predict a tilted orientation for the helical region of dynorphin A(1-17) in dimyristoylphosphatidylcholine bilayers

The structural properties of the endogenous opioid peptide dynorphin A(1-17) (DynA), a potential analgesic, were studied with molecular dynamics simulations in dimyristoylphosphatidylcholine bilayers. Starting with the known NMR structure of the peptide in dodecylphosphocholine micelles, the N-terminal helical segment of DynA (encompassing residues 1-10) was initially inserted in the bilayer in a perpendicular orientation with respect to the membrane plane. Parallel simulations were carried out from two starting structures, systems A and B, that differ by 4 A in the vertical positioning of the peptide helix. The complex consisted of approximately 26,400 atoms (dynorphin + 86 lipids + approximately 5300 waters). After >2 ns of simulation, which included >1 ns of equilibration, the orientation of the helical segment of DynA had undergone a transition from parallel to tilted with respect to the bilayer normal in both the A and B systems. When the helix axis achieved a approximately 50 degrees angle with the bilayer normal, it remained stable for the next 1 ns of simulation. The two simulations with different starting points converged to the same final structure, with the helix inserted in the bilayer throughout the simulations. Analysis shows that the tilted orientation adopted by the N-terminal helix is due to specific interactions of residues in the DynA sequence with phospholipid headgroups, water, and the hydrocarbon chains. Key elements are the "snorkel model"-type interactions of arginine side chains, the stabilization of the N-terminal hydrophobic sequence in the lipid environment, and the specific interactions of the first residue, Tyr. Water penetration within the bilayer is facilitated by the immersed DynA, but it is not uniform around the surface of the helix. Many water molecules surround the arginine side chains, while water penetration near the helical surface formed by hydrophobic residues is negligible. A mechanism of receptor interaction is proposed for DynA, involving the tilted orientation observed from these simulations of the peptide in the lipid bilayer.     

The peptide-substrate-binding domain of collagen prolyl 4-hydroxylases is a tetratricopeptide repeat domain with functional aromatic residues

Collagen prolyl 4-hydroxylases catalyze the formation of 4-hydroxyproline in -X-Pro-Gly-sequences and have an essential role in collagen synthesis. The vertebrate enzymes are alpha2beta2 tetramers in which the catalytic alpha-subunits contain separate peptide-substrate-binding and catalytic domains. We report on the crystal structure of the peptide-substrate-binding domain of the human type I enzyme refined at 2.3 A resolution. It was found to belong to a family of tetratricopeptide repeat domains that are involved in many protein-protein interactions and consist of five alpha-helices forming two tetratricopeptide repeat motifs plus the solvating helix. A prominent feature of its concave surface is a deep groove lined by tyrosines, a putative binding site for proline-rich Tripeptides. Solvent-exposed side chains of three of the tyrosines have a repeat distance similar to that of a poly-L-proline type II helix. The aromatic surface ends at one of the tyrosines, where the groove curves almost 90 degrees away from the linear arrangement of the three tyrosine side chains, possibly inducing a bent conformation in the bound peptide. This finding is consistent with previous suggestions by others that a minimal structural requirement for proline 4-hydroxylation may be a sequence in the poly-L-proline type II conformation followed by a beta-turn in the Pro-Gly segment. Site-directed mutagenesis indicated that none of the tyrosines was critical for tetramer assembly, whereas most of them were critical for the binding of a peptide substrate and inhibitor both to the domain and the alpha2beta2 enzyme tetramer.     

Influence of the hydrophilic face on the folding ability and stability of alpha-helix bundles: relevance to the peptide catalytic activity.

Although not the sole feature responsible, the packing of amino acid side chains in the interior of proteins is known to contribute to protein conformational specificity. While a number of amphipathic peptide sequences with optimized hydrophobic domains has been designed to fold into a desired aggregation state, the contribution of the amino acids located on the hydrophilic side of such peptides to the final packing has not been investigated thoroughly. A set of self-aggregating 18-mer peptides designed previously to adopt a high level of alpha-helical conformation in benign buffer is used here to evaluate the effect of the nature of the amino acids located on the hydrophilic face on the packing of a four alpha-helical bundle. These peptides differ from one another by only one to four amino acid mutations on the hydrophilic face of the helix and share the same hydrophobic core. The secondary and tertiary structures in the presence or absence of denaturants were determined by circular dichroism in the far- and near-UV regions, fluorescence and nuclear magnetic resonance spectroscopy. Significant differences in folding ability, as well as chemical and thermal stabilities, were found between the peptides studied. In particular, surface salt bridges may form which would increase both the stability and extent of the tertiary structure of the peptides. The structural behavior of the peptides may be related to their ability to catalyze the decarboxylation of oxaloacetate, with peptides that have a well-defined tertiary structure acting as true catalysts.     

Interactions of melittin, a preprotein model, with detergents.

Bee venom melittin is a water-soluble tetramer of identical polypeptide chains. Each chain has 26 residues. The 20 N-terminal residues are hydrophobic and the 6 C-terminal residues are basic. Melittin has been shown to integrate into natural and synthetic membranes and to lyse a wide variety of cells. To understand how a water-soluble protein can spontaneously partition into a membrane, we have studied the interaction of melittin with micelles of deoxycholate (DOC), Brij 58, and sodium dodecyl sulfate (NaDodSO4). Circular dichroism spectra showed that NaDodSO4, an ionic detergent, and Brij 58, a nonionic detergent, caused similar major changes in the protein's conformation. Gel filtration studies revealed that melittin forms mixed micelles with either Brij or DOC. The melittin-DOC mixed micelles have 2 mol of DOC per mol of melittin. Cross-linking studies with dimethyl suberimidate confirmed that the protein is a tetramer and showed that it becomes monomeric either in mixed micelles with Brij or DOC or in butanol. Despite this major structural change of melittin in the presence of an amphiphile, the covalently cross-linked form is as active in human erythrocyte lysis as the native protein.     

13C-NMR studies of the membrane structure of enveloped virions (vesicular stomatitis virus).

The mobility of the lipids in the bilayer of the envelope of vesicular stomatitis virus has been probed over its complete space by the biosynthetic incorporation of [N-13CH3]- choline as a probe for the polar head groups and [3-13C]- and [11-13C] oleic acid and [16-13C]- palmitic acid for the hydrophobic region of the bilayer. These precursors were effectively incorporated as established by the concomitant administration of the same precursors in radioactive form. Spin lattice relaxation time measurements (T1) of the 13C enriched segments in complete virus envelope allowed estimation of their mobility. The mobility of the polar head groups is restricted, probably due to ionic interactions with neighbouring acidic phospholipids (phosphatidylserine) and/or acidic side chains of the glycoprotein (G-protein). The rigidity of the hydrophobic part of the bilayer is due to the high cholesterol content and interaction with the immersing polypeptide chains of the G- and possibly M-protein. The rigidity is limited to a depth of about 15 A ranging from the inner and outer surface, whereas the inner core of the bilayer is fluid. Tryptic cleavage of the hydrophilic part of the G-protein allows the lipophilic immersing polypeptide fragment to enter further the bilayer which then reduces the fluidity of the hydrocarbon chains in the core region by lipid-protein interactions.     

Membrane insertion of a voltage sensor helix

Most membrane proteins contain a transmembrane (TM) domain made up of a bundle of lipid-bilayer-spanning α-helices. TM α-helices are generally composed of a core of largely hydrophobic amino acids, with basic and aromatic amino acids at each end of the helix forming interactions with the lipid headgroups and water. In contrast, the S4 helix of ion channel voltage sensor (VS) domains contains four or five basic (largely arginine) side chains along its length and yet adopts a TM orientation as part of an independently stable VS domain. Multiscale molecular dynamics simulations are used to explore how a charged TM S4 α-helix may be stabilized in a lipid bilayer, which is of relevance in the context of mechanisms of translocon-mediated insertion of S4. Free-energy profiles for insertion of the S4 helix into a phospholipid bilayer suggest that it is thermodynamically favorable for S4 to insert from water to the center of the membrane, where the helix adopts a TM orientation. This is consistent with crystal structures of Kv channels, biophysical studies of isolated VS domains in lipid bilayers, and studies of translocon-mediated S4 helix insertion. Decomposition of the free-energy profiles reveals the underlying physical basis for TM stability, whereby the preference of the hydrophobic residues of S4 to enter the bilayer dominates over the free-energy penalty for inserting charged residues, accompanied by local distortion of the bilayer and penetration of waters. We show that the unique combination of charged and hydrophobic residues in S4 allows it to insert stably into the membrane.     

Antiparallel four-stranded coiled coil specified by a 3-3-1 hydrophobic heptad repeat

Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.