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BBA - Biomembranes (v.1818, #1)

Reviewer Acknowledgment (pp. iii-vi).

New Title Page (pp. i).

Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs by Abul Kalam Azad; Naoki Yoshikawa; Takahiro Ishikawa; Yoshihiro Sawa; Hitoshi Shibata (pp. 1-11).

Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip ( Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H₂O₂. TgTIP1;1- and TgTIP1;2-mediated H₂O₂ conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H₂O₂ conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.► Isolation and structural characterization of two TIP genes from tulip petals. ► Construction of TIP mutants to mimic the ar/R constrictions of 9 TIPs in Arabidopsis. ► Building of 3D models and analysis of pore conformations of TIP water channels. ► Expression and functional analysis of wild and mutant TIP homologs. ► Amino acid at LE2 position in ar/R constriction determines the substrate selectivity

Keywords: Abbreviations; ar/R; aromatic/arginine; H; ₂; DCFDA; 2,7-dichlorodihydrofluorescein diacetate; NPA; Asn-Pro-Ala; PIP; plasma membrane intrinsic protein; PM; plasma membrane; RACE; rapid amplification of cDNA ends; TIP; tonoplast intrinsic protein; TM; transmembrane; VM; vacuolar membranes; WCA; water channel activity; YNB; yeast nitrogen baseAquaporin; ar/R selectivity filter; Transport selectivity; TgTIP; TIP homolog; Water channel activity

Caveolin-1 hydrophobic segment peptides insertion into membrane mimetic systems: Role of Proline residue by Satoko Aoki; Richard M. Epand (pp. 12-18).

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103–122 that are anchored to the membrane with cholesterol-rich domains. Previously, we reported that changing the Pro¹¹⁰ residue to Ala (the P110A mutant) prevents not only the localization of the protein into lipid rafts but also the formation and functioning of caveolae. The conformational state of caveolin-1 can be shifted toward the transmembrane arrangement by this single amino acid mutation. To model the conformation, and extent of membrane insertion of this segment into membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of caveolin-1 having the sequence KKKKLSTIFGIPMALIWGIYFAILKKKKK-amide and the mutated version, KKKKLSTIFGIAMALIWGIYFAILKKKKK-amide. These peptides contain flanking Lys residues to facilitate purification and handling of the peptide. Circular dichroism measurements demonstrated that the mutated peptide has increased helical content compared with the wild type both in the presence and absence of lipid. The fluorescence emission from the Trp residues in the peptide showed significant blue shifts in the presence of liposomes, however the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. Our overall findings support our studies with the intact protein in cells and suggest that the peptide of WT caveolin-1 hydrophobic segment has an intrinsic preference not to maintain its conformation as a rigid transmembrane helix. Substituting the Pro residue with an Ala allows the peptide to exist in a more hydrophobic environment likely as a consequence of a change in its conformation to a straight hydrophobic helix that traverses the membrane.► Hydrophobic segment of caveolin inserts into membranes. ► Single residue substitution allows greater membrane penetration of peptide. ► Properties of isolated peptide correspond to that of protein. ► Cholesterol inhibits membrane insertion.

Keywords: Abbreviations; DPPC; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; F; ₀; maximum fluorescence emission intensity without quencher; F; and maximum fluorescence emission intensity with the quencher; K; _SV; Stern–Volmer quenching constant; LPC; Lysophosphatidylcholine (the 1-palmitoyl form); P110A; the peptide KKKKLSTIFGIAMALIWGIYFAILKKKKK-amide; PO/DP/Ch; equimolar mixture of POPC, DPPC and cholesterol; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; SUV; small unilamellar vesicle; WT; the peptide KKKKLSTIFGIPMALIWGIYFAILKKKKK-amideCaveolin-1; Hydrophobic segment; Monotopic protein; Re-entrant helix; Transmembrane helix

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: Implications for interpreting phase behavior by Janos Juhasz; James H. Davis; Frances J. Sharom (pp. 19-26).

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (l_d) domains on the surface of GUVs composed of 40:60mol% DOPC/DPPC, over a wide range of temperatures (14–25°C). The fluorescent lipid, 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of Lissamine^TM rhodamine B 1,2-dihexadecanoyl- sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.Display Omitted► The l_d-gel phase transition of DOPC/DPPC phospholipid mixtures was studied in GUVs. ► Confocal fluorescence microscopy revealed gel phase patch and fibril domains. ► All tested fluorescent probes were excluded from patch domains. ► NBD-DPPE partitioned very strongly into fibril domains. ► No fibril domains were detected using Rh-DPPE, which affected probe partitioning.

Keywords: Abbreviations; Bodipy-PC; 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-; s; -indacene-3-pentanoyl)-1-hexadecanoyl-; sn; -glycero-3-phosphocholine; CFM; confocal fluorescence microscopy; DiD; 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine 4-chlorobenzenesulfonate salt; DiIC; ₁₈; 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; DLPC; 1,2-dilauroyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; GUV; giant unilamellar vesicle; ITO; indium tin oxide; MLVs; multilamellar vesicles; NBD-DPPE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); NMR; nuclear resonance spectroscopy; POPC; 1-palmitoyl-2-oleoly-; sn; -glycerophosphocholine; Rh-DPPE; Lissamine; ^TM; rhodamine B 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine; TR-DPPE; Texas Red 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolaminePhase transition; Giant unilamellar vesicle; Fluorescent probe; Confocal fluorescence microscopy; Partitioning; Patch and fibril domains

Alterations of the C-terminal end do not affect in vitro or in vivo activity of surfactant protein C analogs by Almlen Andreas Almlén; Guy Vandenbussche; Bim Linderholm; Haegerstrand-Bjorkman Marie Haegerstrand-Björkman; Jan Johansson; Tore Curstedt (pp. 27-32).

The secondary structure, orientation and hydrogen/deuterium exchange of SP-C33, a surfactant protein C analog, in 1,2-dipalmitoyl- sn-glycero-3-phosphocholine/egg phosphatidylglycerol (8:2, wt./wt.) bilayers, was studied by attenuated total reflection Fourier transform infrared spectroscopy. This showed a transmembrane α-helix, in which about 55% of the amide hydrogens do not exchange for up to 20h. Moreover, C-terminally modified SP-C33, either truncated after position 30, or having the methionine at position 31 exchanged for either lysine or isoleucine, showed the same secondary structure and orientation. The different peptides, suspended in 1,2-dipalmitoyl- sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol 68:31 (wt./wt.), were tested for surfactant activity in vitro in a captive bubble surfactometer and in vivo in an animal model of respiratory distress syndrome using premature rabbit fetuses. All preparations showed similar surface activity in the captive bubble surfactometer. Also, in the rabbit model, all preparations performed equally well and significantly better than non-treated controls, both regarding tidal volumes and lung gas volumes. Thus, truncation or residue replacements in the C-terminal part of SP-C33 do not seem to affect membrane association or surfactant activity.► Secondary structure and orientation of surfactant protein C analog SP-C33 in a DPPC membrane is equal to that of native SP-C. ► C-terminal modifications of SP-C33 do not alter its surfactant activity. ► C-terminal truncation of SP-C33 does not affect its surfactant activity.

Keywords: Abbreviations; CBS; captive bubble surfactometer; DPPC; 1,2-dipalmitoyl-; sn; -glycero-phosphocholine; ATR-FTIR; attenuated total reflection Fourier transform infrared spectroscopy; LGV; lung gas volume; PEEP; positive end expiratory pressure; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerol; RDS; respiratory distress syndrome; SP; surfactant proteinPulmonary surfactant; Respiratory distress syndrome; Membrane protein; Synthetic peptide

Identification and functional characterization of zebrafish K_2P10.1 (TREK2) two-pore-domain K⁺ channels by Jakob Gierten; David Hassel; Patrick A. Schweizer; Rüdiger Becker; Hugo A. Katus; Dierk Thomas (pp. 33-41).

Two-pore-domain potassium (K_2P) channels mediate K⁺ background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K_2P currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K_2P channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K_2P10.1 channel. K_2P10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK_2P10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K_2P10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K_2P10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K⁺ selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK_2P10.1, induced robust activation of zK_2P10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK_2P10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K_2P10.1 two-pore-domain K⁺ channels that exhibit structural and functional properties largely similar to human K_2P10.1. We conclude that the zebrafish represents a valid model to study K_2P10.1 function in vivo.►The zebrafish genome harbors a family K_2P potassium channels. ►K_2P10.1 (TREK2) was cloned from zebrafish cDNA. ►Human and zebrafish K_2P10.1 exhibit similar ion selectivity and modulation. ►hK_2P10.1 and zK_2P10.1 are targeted by common antiarrhythmic drugs. ►Zebrafish may serve as a model to study the role of K_2P in physiology and disease

Keywords: Abbreviations; AA; arachidonic acid; aa; amino acids; CDS; coding sequence; E; _rev; reversal potential; h; human; I; current; I; _KP; cardiac plateau current; ISH; in situ hybridization; K; _2P; channel; two-pore-domain potassium channel; M1 and M2; first and second translation initiation sites; ORF; open reading frame; P; pore domain; PA; palmitic acid; PKC; protein kinase C; PMA; phorbol 12-myristate 13-acetate; PUFA; polyunsaturated acid; r; rat; RMP; resting membrane potential; TASK; TWIK-related acid sensitive K; ⁺; channel; TM; transmembrane domain; TRAAK; TWIK-related arachidonic acid-stimulated K; ⁺; channel; TREK; TWIK-related K; ⁺; channel; TWIK; tandem of P domains in a weak inward rectifying K; ⁺; channel; UTR; untranslated region; V; voltage; z; zebrafishElectrophysiology; Ion channel; K; ⁺; leak current; K; _2P; 10.1 (TREK2); Membrane potential; Zebrafish

Direct in vivo interaction of the antibiotic primycin with the plasma membrane of Candida albicans: An EPR study by Virag Eszter Virág; Joseph Belagyi; Zoltán Gazdag; Vagvolgyi Csaba Vágvölgyi; Miklós Pesti (pp. 42-48).

The direct interaction of the antibiotic primycin with the plasma membrane was investigated by employing the well-characterized ergosterol-producing, amphotericin B-sensitive parental Candida albicans strain 33erg⁺ and its ergosterol-less amphotericin B-resistant plasma membrane mutant erg-2. The growth inhibition concentration in shaken liquid medium was 64μgml⁻¹ for 33erg⁺ and 128μgml⁻¹ for erg-2, suggesting that the plasma membrane composition influences the mode of action of primycin. To determine the primycin-induced changes in the plasma membrane dynamic, electron paramagnetic resonance (EPR) spectroscopy methods were used, the spin-labeled fatty acid 5-(4,4-dimethyloxazolidine- N-oxyl)stearic acid) being applied for the in vivo measurements. The phase transition temperatures of untreated strain 33erg⁺ and its mutant erg-2 were 12.5°C and 11°C, respectively. After 128μgml⁻¹ primycin treatment, these values increased to 17.5°C and 16°C, revealing a significant reduction in the phospholipid flexibility. Saturation transfer EPR measurements demonstrated that, the rotational correlation times of the spin label molecule for the control samples of 33erg⁺ and erg-2 were 60ns and 100ns. These correlation times gradually decreased on the addition of increasing primycin concentrations, reaching 8μs and 1μs. The results indicate the plasma membrane “rigidizing” effect of primycin, a feature that may stem from its ability to undergo complex formation with membrane constituent fatty acid molecules, causing alterations in the structures of phospholipids in the hydrophobic surface near the fatty acid chain region.► The antibiotic primycin as a membrane destructive agent. ► Primycin induced an increase of phase transition of membranes. ► Primycin caused slower molecular motions in membranes. ► Biological membranes divided into two distinct phase as a consequence of primycin. ► Fatty acids as the main target of primycin.

Keywords: Abbreviations; AmB; amphotericin B; C. albicans; Candida albicans; 5-SASL; 5-(4,4-dimethyloxazolidine-; N; -oxyl)stearic acid; EPR spectroscopy; electron paramagnetic resonance spectroscopy; G; Gauss; ST-EPR spectroscopy; saturation transfer electron paramagnetic resonance spectroscopy Candida albicans; EPR; Membrane fluidity; Primycin; ST-EPR; 5-SASL

Structural basis for misfolding at a disease phenotypic position in CFTR: Comparison of TM3/4 helix-loop-helix constructs with TM4 peptides by Cory M. Mulvihill; Charles M. Deber (pp. 49-54).

Understanding the residue-dependent effects of disease-phenotypic mutations in multi-spanning membrane proteins is an essential step toward the development of corrective therapies. As a systematic approach to further elucidate mutant-dependent mis-folding consequences, we prepared two libraries: one consisting of 20 helix-loop-helix (“hairpin”) constructs derived from helices 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR) (residues 194–241) in which the CF-phenotypic position Val-232 was substituted individually to each of the 20 commonly-occurring amino acids; and a second library consisting of 20 single-stranded TM4 peptides (CFTR residues 221–241) similarly substituted at position 232. Both libraries were analyzed to measure mutant-dependent variations in mobility on SDS-PAGE; size and shape on size exclusion chromatography; retention times on reverse phase HPLC; and helical content by circular dichroism spectroscopy. Analysis of a scatter plot between TM3/4 hairpin and TM4 peptide retention times showed a strong correlation (r=0.94, p<0.05), with retention times largely a function of residue hydrophobicity. In contrast, while the hairpin library migrated over a significant range on SDS-PAGE, migration rates for TM4 hydrophobic residues at position 232 converged at a single value, suggesting that residue-dependent re-orientations of hairpin van der Waals interfaces may expose varying faces of the TM3 and/or TM4 helices to the SDS detergent. The overall results suggest that mutant-mediated variations are a principal determinant of tertiary interhelical folding interactions in membranes.► A CF-phenotypic position in a helical hairpin is studied using all 20 amino acids. ► Residue hydrophobicity is the dominant effect on protein–detergent interactions. ► Mutant-mediated variations determine the extent of folding of a given construct. ► Our results may portend the structural consequences in a natural lipid bilayer.

Keywords: Abbreviations; CD; circular dichroism; CFTR; human cystic fibrosis transmembrane conductance regulator; ESI-MS; electron spray ionization-mass spectrometry; HPLC; high performance liquid chromatography; MALDI; matrix assisted laser desorption ionization-mass spectrometry; MES; 2-(; N; -morpholino)ethanesulfonic acid; MRE; mean residue ellipticity; RP; reverse phase; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC; size exclusion chromatography; TFA; trifluoroacetic acid; TM; transmembraneCystic fibrosis transmembrane conductance regulator (CFTR); Hydropathy; Membrane protein; SDS-PAGE

Electrostatic interaction between inactivation ball and T1–S1 linker region of Kv1.4 channel by Zhuo Fan; Xuying Ji; Mingyu Fu; Wanming Zhang; Du Zhang; Zhongju Xiao (pp. 55-63).

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a “ball and chain” inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the “ball and chain” inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1–S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1–S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.► Mutating negatively charged residues on T1–S1 linker to opposite polar slowed inactivation of Kv1.4 moderately. ► Mutating positively charged residues of inactivation ball to opposite polar slowed inactivation of Kv1.4 strongly. ► Mutating charged residues of both inactivation ball and T1–S1 linker slowed the inactivation compared with Kv1.4WT. ► The voltage-dependence of inactivation was changed, whereas the voltage-dependence of activation was not changed. ► Inserting negatively charged peptide in different positions of inactivation domain slowed the inactivation rate.

Keywords: Kv1.4 channel; Inactivation; “Ball and chain” model; Electrostatic interaction

A consensus segment in the M2 domain of the hP2X₇ receptor shows ion channel activity in planar lipid bilayers and in biological membranes by Cristina Alves Magalhães de Souza; Pedro Celso Nogueira Teixeira; Robson Xavier Faria; Oxana Krylova; Peter Pohl; Luiz Anastacio Alves (pp. 64-71).

The P2X₇ receptor (P2X₇R) is an ATP-gated, cation-selective channel permeable to Na⁺, K⁺ and Ca²⁺. This channel has also been associated with the opening of a non-selective pore that allows the flow of large organic ions. However, the biophysical properties of the P2X₇R have yet to be characterized unequivocally. We investigated a region named ADSEG, which is conserved among all subtypes of P2X receptors (P2XRs). It is located in the M2 domain of hP2X₇R, which aligns with the H5 signature sequence of potassium channels. We investigated the channel forming ability of ADSEG in artificial planar lipid bilayers and in biological membranes using the cell-attached patch-clamp techniques. ADSEG forms channels, which exhibit a preference for cations. They are voltage independent and show long-term stability in planar lipid bilayers as well as under patch-clamping conditions. The open probability of the ADSEG was similar to that of native P2X₇R. The conserved part of the M2 domain of P2X₇R forms ionic channels in planar lipid bilayers and in biological membranes. Its electrophysiological characteristics are similar to those of the whole receptor. Conserved and hydrophobic part of the M2 domain forms ion channels.► ADSEG peptide forms channels in planar lipid bilayer and patch-clamp. ► Single channel conductance of monovalent and divalent cations: K⁺, Na⁺, Ca⁺⁺, Mg⁺⁺ and NMDG. ► Open probability channel of ADSEG in planar lipid bilayer and patch-clamp. ► Planar lipid system stability over 120min.

Keywords: P2X; ₇; receptor; Ionic channel activity; Peptide single channel activity; Artificial planar lipid bilayer; Patch-clamp

Membrane topology screen of secondary transport proteins in structural class ST[3] of the MemGen classification. Confirmation and structural diversity by Ramon ter Horst; Juke S. Lolkema (pp. 72-81).

The MemGen structural classification of membrane proteins groups families of proteins by hydropathy profile alignment. Class ST[3] of the MemGen classification contains 32 families of transporter proteins including the IT superfamily. Transporters from 19 different families in class ST[3] were evaluated by the TopScreen experimental topology screening method to verify the structural classification by MemGen. TopScreen involves the determination of the cellular disposition of three sites in the polypeptide chain of the proteins which allows for discrimination between different topology models. For nearly all transporters at least one of the predicted localizations is different in the models produced by MemGen and predictor TMHMM. Comparison to the experimental data showed that in all cases the prediction by MemGen was correct. It is concluded that the structural model available for transporters of the [st324]ESS and [st326]2HCT families is also valid for the other families in class ST[3]. The core structure of the model consists of two homologous domains, each containing 5 transmembrane segments, which have an opposite orientation in the membrane. A reentrant loop is present in between the 4th and 5th segments in each domain. Nearly all of the identified and experimentally confirmed structural variations involve additions of transmembrane segments at the boundaries of the core model, at the N- and C-termini or in between the two domains. Most remarkable is a domain swap in two subfamilies of the [st312]NHAC family that results in an inverted orientation of the proteins in the membrane.► Experimental validation of the fold of 32 transporter families. ► Core structure of ion transporter superfamily. ► Structural diversity of class ST[3] transporters. ► Additional TMSs found at N- and C-termini and at domain interface.

Keywords: Abbreviations; LIC; ligation independent cloning; GFP; green fluorescent protein; FM; fluorescein-5-maleimide; TMS; transmembrane segmentMembrane topology; Structural classification; ST[3]; Topology prediction; MemGen; Reporter fusion

The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems by Luis Fernando Reyes; Thatyane M. Nobre; Felippe J. Pavinatto; Maria E.D. Zaniquelli; Luciano Caseli; Osvaldo N. Oliveira Jr.; Araujo Ana Paula U. Araújo (pp. 82-89).

Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244, 239 and 236 residues (rPAC²⁴⁴, rPAC²³⁹ and rPAC²³⁶), were studied. rPAC had the strongest interaction with the DPPG monolayer, inducing a large expansion in its surface pressure–area isotherm. The affinity to DPPG decreased with increased deletion of the C-terminal region. When the C-terminal region was deleted completely (rPAC²³⁶), the interaction was recovered, probably because other hydrophobic regions were exposed to the membrane. Using Polarization Modulated-Infrared Reflection Absorption Spectroscopy (PM-IRRAS) we observed that at a bare air/water interface rPAC comprised mainly α-helix structures, the C-terminal region had unordered structures when interacting with DPPG. For rPAC²³⁶ the α-helices were preserved even in the presence of DPPG. These results confirm the importance of the C-terminal region for PAC-ER membrane interaction. The partial unfolding only with preserved C-terminal appears a key step for the protein to reach the cytosol and develop its toxic activity.► We investigated the interaction between recombinant PAC and Langmuir monolayers. ► Three catalytically active, truncated PACs with increasing deletion of the C-terminal region were studied. ► The affinity to DPPG decreased with increased deletion of the C-terminal region. ► Using PM-IRRAS we confirm the importance of the C-terminal region for PAC-membrane interaction. ► We consider this interaction fundamental for the protein to reach the cytosol and develop its toxic activity.

Keywords: Ribosome inactivating protein; Langmuir monolayers; Pulchellin; Membrane interaction; PM-IRRAS

Probing the interaction of lipids with the non-annular binding sites of the potassium channel KcsA by magic-angle spinning NMR by Phedra Marius; Maurits R.R. de Planque; Philip T.F. Williamson (pp. 90-96).

The activity of the potassium channel KcsA is tightly regulated through the interactions of anionic lipids with high-affinity non-annular lipid binding sites located at the interface between the channel's subunits. Here we present solid-state phosphorous NMR studies that resolve the negatively charged lipid phosphatidylglycerol within the non-annular lipid-binding site. Perturbations in chemical shift observed upon the binding of phosphatidylglycerol are indicative of the interaction of positively charged sidechains within the non-annular binding site and the negatively charged lipid headgroup. Site directed mutagenesis studies have attributed these charge interactions to R64 and R89. Functionally the removal of the positive charges from R64 and R89 appears to act synergistically to reduce the probability of channel opening.Display Omitted► Solid-state 31P MAS NMR studies have been used to characterise binding to the non-annular binding site of KcsA. ► Residues R64 and R89 have been identified as important in the recognition of anionic lipids in the non-annular binding site. ► Single-channel current recordings demonstrate the functional importance of these interactions for KcsA activity.

Keywords: Ion channel; Solid-state NMR; Lipid/protein interface; Phosphorous NMR; Magic-angle spinning; Potassium channel KcsA

Cys²⁹⁴ is essential for the function of the human sodium-dependent multivitamin transporter by Abhisek Ghosal; Hamid M. Said (pp. 97-102).

The sodium-dependent multivitamin transporter (SMVT) plays an important role in biotin uptake in the intestine and other cell types. While significant knowledge has been gained with regard to regulation and cell biology of the SMVT system, there is little known about its structure-function relationships. Here we examined the role of each of the ten conserved (among species) cysteine residues in the function of the human SMVT (hSMVT) using site-directed mutagenesis. Our results showed a significant impairment in biotin uptake only in cells transfected with hSMVT mutated at Cys²⁹⁴, but not at the other conserved cysteine residues; the impairment in biotin uptake caused by mutating Cys²⁹⁴ was not related to the polar status of substituting amino acid. The inhibition in hSMVT function upon mutating Cys²⁹⁴ was mediated via a significant reduction in the V_max, but not the apparent K_m, of the biotin uptake process, suggesting a decrease in the number (and/or activity) of hSMVT but not affinity. Biotinylation assay confirmed this suggestion by showing a marked reduction in the level of expression of the mutated protein at the cell membrane, without affecting total cellular level of induced hSMVT. These results show an important role for Cys²⁹⁴ in the function and cell biology of hSMVT.► A significant inhibition in biotin uptake occurs upon mutating Cys²⁹⁴. ► The effect is not related to polar status of substituted cysteine residue. ► The inhibition is mediated via significant decrease in V_max of the biotin uptake. ► Mutating Cys²⁹⁴ leads to a marked reduction in surface expression of hSMVT.

Keywords: hSMVT; Cysteine residue; Biotin uptake

Effect of surfactant concentration, compression ratio and compression rate on the surface activity and dynamic properties of a lung surfactant by Sameh M.I. Saad; Zdenka Policova; Edgar J. Acosta; A. Wilhelm Neumann (pp. 103-116).

This paper reports dynamic surface tension experiments of a lung surfactant preparation, BLES, for a wide range of concentrations, compression ratios and compression rates. These experiments were performed using Axisymmetric Drop Shape Analysis–Constrained Sessile Drop (ADSA–CSD). The main purpose of the paper is to interpret the results in terms of physical parameters using the recently developed Compression–Relaxation Model (CRM). In the past, only the minimum surface tension was used generally for the characterization of lung surfactant films; however, this minimum value is not a physical parameter and depends on the compression protocol. CRM is based on the assumption that the dynamic surface tension response is governed by surface elasticities, adsorption and desorption of components of the lung surfactant. The ability of CRM to fit the surface tension response closely for a wide variety of parameters (compression ratio, compression rate and surfactant concentration) and produce sensible values for the elastic and kinetic parameters supports the validity of CRM.► ADSA–CSD measures dynamic surface tension of lung surfactants at relevant conditions. ► Results are analyzed using a recently developed Compression-Relaxation Model (CRM). ► The sensitivity of the model is evaluated at different conditions. ► Effect of concentration on the four dynamic parameters is minimal beyond 8mg/ml.

Keywords: Pulmonary surfactant; Compression Relaxation Model; ADSA–CSD; Relaxation; Adsorption; Elasticity

Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C by Muller Alexandra Müller; Kloppel Christine Klöppel; Megan Smith-Valentine; Judith Van Houten; Martin Simon (pp. 117-124).

Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content.► The release of different GPI-anchored proteins from the cell surface membrane is characterized in vitro and in vivo. ► GPI-anchors are cleaved and released into the cellular medium by phospholipase C. ► Phospholipase C isoforms discriminate between different GPI-proteins. ► Our data indicates that release of GPI-proteins by phospholipase C is fast, programmed and selective.

Keywords: Abbreviations; GPI; glycosylphosphatidylinositol; PLC; phospholipase C; PI-PLC; phosphatidylinositol specific phospholipase C; GPI-PLC; glycosylphosphatidylinositol specific phospholipase C; CRD; cross-reacting determinant; ELISA; enzyme linked immunosorbent assay; HM; high molecular; SM; small molecular; DAF; decay accelerating factor; GAPDH; glycerinealdehyde-3-dehydrogenaseGPI-anchor; Phospholipase C; Surface antigen; Antigenic variation; GPI-PLC; GPI-release

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BBA - Biomembranes (v.1818, #1)

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BBA - Biomembranes (v.1818, #1)