ERK5 signalling pathway is a novel target of sorafenib: Implication in EGF biology.

Sorafenib is a multikinase inhibitor widely used in cancer therapy with an antitumour effect related to biological processes as proliferation, migration or invasion, among others. Initially designed as a Raf inhibitor, Sorafenib was later shown to also block key molecules in tumour progression such as VEGFR and PDGFR. In addition, sorafenib has been connected with key signalling pathways in cancer such as EGFR/EGF. However, no definitive clue about the molecular mechanism linking sorafenib and EGF signalling pathway has been established so far. Our data in HeLa, U2OS, A549 and HEK293T cells, based on in silico, chemical and genetic approaches demonstrate that the MEK5/ERK5 signalling pathway is a novel target of sorafenib. In addition, our data show how sorafenib is able to block MEK5-dependent phosphorylation of ERK5 in the Ser218/Tyr220, affecting the transcriptional activation associated with ERK5. Moreover, we demonstrate that some of the effects of this kinase inhibitor onto EGF biological responses, such as progression through cell cycle or migration, are mediated through the effect exerted onto ERK5 signalling pathway. Therefore, our observations describe a novel target of sorafenib, the ERK5 signalling pathway, and establish new mechanistic insights for the antitumour effect of this multikinase inhibitor.

Location, Orientation and Aggregation of Bardoxolone-ME, CDDO-ME, in a Complex Phospholipid Bilayer Membrane.

Bardoxolone methyl (CDDO-Me), a synthetic derivative of the naturally occurring triterpenoid oleanolic acid, displays strong antioxidant, anticancer and anti-inflammatory activities, according to different bibliographical sources. However, the understanding of its molecular mechanism is missing. Furthermore, CDDO-Me has displayed a significant cytotoxicity against various types of cancer cells. CDDO-Me has a noticeable hydrophobic character and several of its effects could be attributed to its ability to be incorporated inside the biological membrane and therefore modify its structure and specifically interact with its components. In this study, we have used full-atom molecular dynamics to determine the location, orientation and interactions of CDDO-Me in phospholipid model membranes. Our results support the location of CDDO-Me in the middle of the membrane, it specifically orients so that the cyano group lean towards the phospholipid interface and it specifically interacts with particular phospholipids. Significantly, in the membrane the CDDO-Me molecules specifically interact with POPE and POPS. Moreover, CDDO-Me does not aggregates in the membrane but it forms a complex conglomerate in solution. The formation of a complex aggregate in solution might hamper its biological activity and therefore it should be taken into account when intended to be used in clinical assays. This work should aid in the development of these molecules opening new avenues for future therapeutic developments.

Multibeam Reflectarrays in Ka-Band for Efficient Antenna Farms Onboard Broadband Communication Satellites.

Broadband communication satellites in Ka-band commonly use four reflector antennas to generate a multispot coverage. In this paper, four different multibeam antenna farms are proposed to generate the complete multispot coverage using only two multibeam reflectarrays, making it possible to halve the number of required antennas onboard the satellite. The proposed solutions include flat and curved reflectarrays with single or dual band operation, the operating principles of which have been experimentally validated. The designed multibeam reflectarrays for each antenna farm have been analyzed to evaluate their agreement with the antenna requirements for real satellite scenarios in Ka-band. The results show that the proposed configurations have the potential to reduce the number of antennas and feed-chains onboard the satellite, from four reflectors to two reflectarrays, enabling a significant reduction in cost, mass, and volume of the payload, which provides a considerable benefit for satellite operators.

Chromatin immunoprecipitation and high throughput sequencing of SVCV-infected zebrafish reveals novel epigenetic histone methylation patterns involved in antiviral immune response.

Chromatin immunoprecipitation (ChIP) and high throughput sequencing (ChIP-seq) have been used to assess histone methylation (epigenetic modification) dynamics within the internal organs of zebrafish after spring viremia of carp virus (SVCV) infection. Our results show H3K4me3 up-methylation in gene promoters associated with innate immune response during the first 5 days after SVCV infection. Gene Ontology (GO) enrichment analysis confirmed up-methylation in 218 genes in the "immune system process" category. In particular, the promoters of interferon (ifn), interferon stimulated genes (isg), Toll-like receptors (tlr) and c-reactive protein (crp) multi gene sets were marked with the permissive H3K4 methylation. Higher histone 3 methylation was associated with higher transcription levels of the corresponding genes. Therefore, the evidence presented here suggests that transcriptional regulation at the promoter level of key immune genes of the interferon signaling pathway and c-reactive proteins genes can be modulated by epigenetic modification of histones. This study emphasizes the importance of epigenetic control in the response of zebrafish to SVCV infection.

Dual-Polarization Ku-Band Compact Spaceborne Antenna Based on Dual-Reflectarray Optics.

This article demonstrated an accurate analysis technique for dual-reflectarray antennas that take into account the angle of incidence of the impinging electric field on the main reflectarray cells. The reflected field on the sub and the main reflectarray surfaces is computed using Method of Moments in the spectral domain and assuming local periodicity. The sub-reflectarray is divided into groups of elements and the field radiated by each group is used to compute the incident and reflected field on the main reflectarray cells. A 50-cm demonstrator in Ku-band that provides European coverage has been designed, manufactured and tested to validate the analysis technique. The measured radiation patterns match the simulations and they fulfill the coverage requirements, achieving a cross-polar discrimination better than 25 dB in the frequency range: 12.975-14.25 GHz.

Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA).

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp(67)-Glu(71)-Asp(80) inactivation triad within the channel structure and its bearing on the selectivity filter.

Partitioning of liquid-ordered/liquid-disordered membrane microdomains induced by the fluidifying effect of 2-hydroxylated fatty acid derivatives.

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses.

Detergent-labile, supramolecular assemblies of KcsA: relative abundance and interactions involved.

In this work, we illustrate the ability of the prokaryotic potassium channel KcsA to assemble into a variety of supramolecular clusters of defined sizes containing the tetrameric KcsA as the repeating unit. Such clusters, particularly the larger ones, are markedly detergent-labile and thus, disassemble readily upon exposure to the detergents commonly used in protein purification or conventional electrophoresis analysis. This is a reversible process, as cluster re-assembly occurs upon detergent removal and without the need of added membrane lipids. Interestingly, the dimeric ensemble between two tetrameric KcsA molecules are quite resistant to detergent disassembly to individual KcsA tetramers and along with the latter, are likely the basic building blocks through which the larger clusters are organized. As to the proteins domains involved in clustering, we have observed disassembly of KcsA clusters by SDS-like alkyl sulfates. As these amphiphiles bind to inter-subunit, "non-annular" sites on the protein, these observations suggest that such sites also mediate channel-channel interactions leading to cluster assembly.

Mutation of Ser-50 and Cys-66 in Snapin modulates protein structure and stability.

Snapin is a 15 kDa protein present in neuronal and non-neuronal cells that has been implicated in the regulation of exocytosis and endocytosis. Protein kinase A (PKA) phosphorylates Snapin at Ser-50, modulating its function. Likewise, mutation of Cys-66, which mediates protein dimerization, impairs its cellular activity. Here, we have investigated the impact of mutating these two positions on protein oligomerization, structure, and thermal stability, along with the interaction with SNARE proteins. We found that recombinant purified Snapin in solution appears mainly as dimers in equilibrium with tetramers. The protein exhibits modest secondary structure elements and notable thermal stability. Mutation of Cys-66 to Ser abolished subunit dimerization, but not higher-order oligomers. This mutant augmented the presence of α-helical structure and slightly increased the protein thermal stability. Similarly, the S50A mutant, mimicking the unphosphorylated protein, also exhibited a higher helical secondary structure content than the wild type, along with greater thermal stability. In contrast, replacement of Ser-50 with Asp (S50D), emulating the protein-phosphorylated state, produced a loss of α-helical structure, concomitant with a decrease in protein thermal stability. In vitro, the wild type and mutants weakly interacted with SNAP-25 and the reconstituted SNARE complex, although S50D exhibited the strongest binding to the SNARE complex, consistent with the observed higher cellular activity of PKA-phosphorylated Snapin. Our observations suggest that the stronger binding of S50D to SNAREs might be due to a destabilization of tetrameric assemblies of Snapin that favor the interaction of protein dimers with the SNARE proteins. Therefore, phosphorylation of Ser-50 has an important impact on the protein structure and stability that appears to underlie its functional modulation.

Bisphenol-S and Bisphenol-F alter mouse pancreatic ß-cell ion channel expression and activity and insulin release through an estrogen receptor ERß mediated pathway.

Bisphenol-S (BPS) and Bisphenol-F (BPF) are current Bisphenol-A (BPA) substitutes. Here we used pancreatic ß-cells from wild type (WT) and estrogen receptor ß (ERß) knockout (BERKO) mice to investigate the effects of BPS and BPF on insulin secretion, and the expression and activity of ion channels involved in ß-cell function. BPS or BPF rapidly increased insulin release and diminished ATP-sensitive K+ (KATP) channel activity. Similarly, 48 h treatment with BPS or BPF enhanced insulin release and decreased the expression of several ion channel subunits in ß-cells from WT mice, yet no effects were observed in cells from BERKO mice. PaPE-1, a ligand designed to preferentially trigger extranuclear-initiated ER pathways, mimicked the effects of bisphenols, suggesting the involvement of extranuclear-initiated ERß pathways. Molecular dynamics simulations indicated differences in ERß ligand-binding domain dimer stabilization and solvation free energy among different bisphenols and PaPE-1. Our data suggest a mode of action involving ERß whose activation alters three key cellular events in ß-cell, namely ion channel expression and activity, and insulin release. These results may help to improve the hazard identification of bisphenols.

The Interaction of Temozolomide with Blood Components Suggests the Potential Use of Human Serum Albumin as a Biomimetic Carrier for the Drug.

The interaction of temozolomide (TMZ) (the main chemotherapeutic agent for brain tumors) with blood components has not been studied at the molecular level to date, even though such information is essential in the design of dosage forms for optimal therapy. This work explores the binding of TMZ to human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP), as well as to blood cell-mimicking membrane systems. Absorption and fluorescence experiments with model membranes indicate that TMZ does not penetrate into the lipid bilayer, but binds to the membrane surface with very low affinity. Fluorescence experiments performed with the plasma proteins suggest that in human plasma, most of the bound TMZ is attached to HSA rather than to AGP. This interaction is moderate and likely mediated by hydrogen-bonding and hydrophobic forces, which increase the hydrolytic stability of the drug. These experiments are supported by docking and molecular dynamics simulations, which reveal that TMZ is mainly inserted in the subdomain IIA of HSA, establishing π-stacking interactions with the tryptophan residue. Considering the overexpression of albumin receptors in tumor cells, our results propose that part of the administered TMZ may reach its target bound to plasma albumin and suggest that HSA-based nanocarriers are suitable candidates for designing biomimetic delivery systems that selectively transport TMZ to tumor cells.

Differential Effects of IGF-1R Small Molecule Tyrosine Kinase Inhibitors BMS-754807 and OSI-906 on Human Cancer Cell Lines.

We have determined the effects of the IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on cell proliferation and cell-cycle phase distribution in human colon, pancreatic carcinoma, and glioblastoma cell lines and primary cultures. IGF-1R signaling was blocked by BMS and OSI at equivalent doses, although both inhibitors exhibited differential antiproliferative effects. In all pancreatic carcinoma cell lines tested, BMS exerted a strong antiproliferative effect, whereas OSI had a minimal effect. Similar results were obtained on glioblastoma primary cultures, where HGUE-GB-15, -16 and -17 displayed resistance to OSI effects, whereas they were inhibited in their proliferation by BMS. Differential effects of BMS and OSI were also observed in colon carcinoma cell lines. Both inhibitors also showed different effects on cell cycle phase distribution, BMS induced G2/M arrest followed by cell death, while OSI induced G1 arrest with no cell death. Both inhibitors also showed different effects on other protein kinases activities. Taken together, our results are indicative that BMS mainly acts through off-target effects exerted on other protein kinases. Given that BMS exhibits a potent antiproliferative effect, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.

The CBS domain protein MJ0729 of Methanocaldococcus jannaschii is a thermostable protein with a pH-dependent self-oligomerization.

CBS domains are small protein motifs, usually associated in tandems, that are involved in binding to adenosyl groups. In humans, several genetic diseases have been associated with mutations in CBS domains, and then, they can be considered as promising targets for the rational design of new drugs. However, there are no structural studies describing their oligomerization states, conformational preferences, and stability. In this work, the oligomerization state, the stability, and conformational properties of the CBS domain protein MJ0729 from Methanocaldococcus jannaschii were explored by using a combination of hydrodynamic (namely, ultracentrifugation, DLS, DOSY-NMR, and gel filtration) and spectroscopic techniques (fluorescence, circular dichroism, NMR, and FTIR). The results indicate that the protein had a pH-dependent oligomerization equilibrium: at pH 7, the dominant species is a dimer, where each monomer is a two-CBS domain protein, and at pH 4.5-4.8, the dominant species is a tetramer, with an oblong shape, as shown by X-ray. Deconvolution of the FTIR spectra indicates that the monomer at physiological pH has 26% alpha-helical structure and 17% beta-sheet, with most of the structure disordered. These results are similar to the percentages of secondary structure of the monomer in the resolved tetrameric X-ray structure (21% of alpha-helical structure and 7% of beta-sheet). At pH 2.5, there was a decrease in the level of secondary structure of the monomer, and formation of intermolecular hydrogen bonds, as shown by FTIR, suggesting the presence of high-molecular weight species. The physiological dimeric species is thermal and chemically very stable with a thermal midpoint of approximately 99 degrees C, as shown by both DSC and FTIR; the GdmCl chemical midpoint of the dimeric species occurs in a single step and was greater than 4 M.

Nicotinic acetylcholine receptor properties are modulated by surrounding lipids: an in vivo study.

In vitro studies carried out on liposomes of defined composition showed that nicotinic acetylcholine receptors (nAChRs) are fully functional when they are reconstituted in a heterogeneous lipid matrix, such as that provided by crude soybean (asolectin [R-Aso]) lipids. However, when they are reconstituted in plain phosphatidylcholine (R-PC) lipids, their functional activity is completely lost (Fong and McNamee, 1986). This kind of study also pointed out that phosphatidic acid (PA) and cholesterol (Chol) play an important role in preserving the ability of this protein to exhibit an optimal channel activity (Fong and McNamee, 1986). Furthermore, it has been shown recently that nAChR, itself, induces the formation of specific PA-rich lipid domains (Poveda et al., 2002). Because Xenopus oocytes incorporate functionally into their plasma membrane nAChRs after intracellular injection of liposomes bearing this protein (Morales et al., 1995), the aim of this work was to determine the effect of the reconstitution lipid matrix on the functional properties of the transplanted nAChRs.

Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids.

Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of cation translocation and Fourier transform-infrared (FT-IR) spectroscopy studies have been used to illustrate the lipid dependence of both AChR function and AChR secondary structure, respectively.

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: a biophysical characterization.

Dihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (-CO-NH-) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-beta-amino acids. This enzyme is an attractive candidate for commercial production of D-aminoacids, which are used in the production of semi-synthetic beta-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH approximately 6.0, which is stable up to pH approximately 9.0, as concluded from fluorescence and CD. Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure.

N-type inactivation of the potassium channel KcsA by the Shaker B "ball" peptide: mapping the inactivating peptide-binding epitope.

The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr(8), which exhibit the strongest STD effects; the C4H in the imidazole ring of His(16); the methyl protons of Val(4), Leu(7), and Leu(10) and the side chain amine protons of one, if not both, the Lys(18) and Lys(19) residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val(4) and Leu(10), whereas His(16) seems similarly affected as before. Conversely, STD effects on Tyr(8) are strongly diminished, and those on Lys(18) and/or Lys(19) are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of (13)C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a beta-hairpin structure when bound to the KcsA channel. Indeed, docking such a beta-hairpin structure into an open pore model for K(+) channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.

Effects of conducting and blocking ions on the structure and stability of the potassium channel KcsA.

This article reports on the interaction of conducting (K(+)) and blocking (Na(+)) monovalent metal ions with detergent-solubilized and lipid-reconstituted forms of the K(+) channel KcsA. Monitoring of the protein intrinsic fluorescence reveals that the two ions bind competitively to KcsA with distinct affinities (dissociation constants for the KcsA.K(+) and KcsA.Na(+) complexes of approximately 8 and 190 mm, respectively) and induce different conformations of the ion-bound protein. The differences in binding affinity as well as the higher K(+) concentration bathing the intracellular mouth of the channel, through which the cations gain access to the protein binding sites, should favor that only KcsA.K(+) complexes are formed under physiological-like conditions. Nevertheless, despite such prediction, it was also found that concentrations of Na(+) well below its dissociation constant and even in the presence of higher K(+) concentrations, cause a remarkable decrease in the protein thermal stability and facilitate thermal dissociation into subunits of the tetrameric KcsA, as concluded from the temperature dependence of the protein infrared spectra and from gel electrophoresis, respectively. These latter observations cannot be explained based on the occupancy of the binding sites from above and suggest that there must be additional ion binding sites, whose occupancy could not be detected by fluorescence and in which the affinity for Na(+) must be higher or at least similar to that of K(+). Moreover, cation binding as reported by means of fluorescence does not suffice to explain the large differences in free energy of stabilization involved in the formation of the KcsA.Na(+) and KcsA.K(+) complexes, which for the most part should arise from synergistic effects of the ion-mediated intersubunit interactions.

Clustering and coupled gating modulate the activity in KcsA, a potassium channel model.

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na(+), and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na(+) blockade. The above functional diversity occurs correlatively to the heterogeneous supramolecular assembly of KcsA into clusters. Clustering of KcsA depends on protein concentration and occurs both in detergent solution and more markedly in reconstituted membranes, including giant liposomes, where some of the clusters are large enough (up to micrometer size) to be observed by confocal microscopy. As in the allosteric conformational spread responses observed in receptor clustering (Bray, D. and Duke, T. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 53-73) our tenet is that physical clustering of KcsA channels is behind the observed multiple coupled gating and diverse functional responses.

Unfolding and refolding in vitro of a tetrameric, alpha-helical membrane protein: the prokaryotic potassium channel KcsA.

2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly alpha-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771-10777]. Here, we report that the effects on the protein structure of increasing concentrations of TFE in detergent solution include two successive protein concentration-dependent, cooperative transitions. In the first of such transitions, occurring at lower TFE concentrations, the tetrameric KcsA simultaneously increases the exposure of tryptophan residues to the solvent, partly loses its secondary structure, and dissociates into its constituent subunits. Under these conditions, simple dilution of the TFE permits a highly efficient refolding and tetramerization of the protein in the detergent solution. Moreover, following reconstitution into asolectin giant liposomes, the refolded protein exhibits nativelike potassium channel activity, as assessed by patch-clamp methods. Conversely, the second cooperative transition occurring at higher TFE concentrations results in the irreversible denaturation of the protein. These results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits, in which folding and oligomerization (or unfolding and dissociation in the other direction of the equilibrium process) are seemingly coupled processes. At higher TFE concentrations this is followed by the irreversible conversion of the unfolded monomers into a denatured protein form.