Check out our New Publishers' Select for Free Articles

BBA - Biomembranes (v.1768, #12)

Editorial Board (pp. ii).

Cumulative Contents (pp. i-xiv).

NMR structural studies on membrane proteins by Ayyalusamy Ramamoorthy (pp. 2947-2948).

Structural characterization of the transmembrane and cytoplasmic domains of human CD4 by Marc Wittlich; Bernd W. Koenig; Silke Hoffmann; Dieter Willbold (pp. 2949-2960).

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly¹⁵N and¹³C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the¹H,¹³C, and¹⁵N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton–proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.

Keywords: Abbreviations; CD4; cluster determinant 4; DPC; dodecylphosphocholine; ER; endoplasmatic reticulum; IMP; integral membrane protein; LB; Luria–Bertani; Lck; lymphocyte specific kinase; MHC II; class II major histocompatibility complex; PCR; polymerase chain reaction; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PFG; pulsed field gradient; RPC; reversed phase chromatography; 2D; two-dimensional; 3D; three-dimensional; TFE; 2,2,2-trifluoroethanol; VpU; viral protein UCD4; HIV-1; VpU; Membrane protein; NMR; CD

A high resolution structure of the putative hinge region in M2 channel-lining segments of the nicotinic acetylcholine receptor by Joanna R. Long; Frank D. Mills; Frank Raucci (pp. 2961-2970).

Transmembrane peptide helices play key roles in signal transduction across cell membranes, yet little is known about their high-resolution structure or the role membrane composition plays in their association, structure, dynamics and ultimately their performance. Using magic angle spinning (MAS) homonuclear dipolar recoupling experiments, the backbone structure at positions L10, L11, and A12 of the M2 ion channel peptide was determined in two lipid systems. Their measurements are in agreement with M2 forming transmembrane helices, but the torsion angles vary considerably from common α−helical values. These measurements show remarkable agreement with a previous computational model of M2 peptides forming a pore domain in which their helices are kinked near the central leucine, L11 [R. Sankararamakrishnan, C. Adcock, M.S.P. Sansom, The pore domain of the nicotinic acetylcholine receptor: Molecular modeling, pore dimensions, and electrostatics, Biophys. J. 71 (1996) 1659–1671]. The generation of high resolution data for transmembrane helices is of critical importance in refining structures for membrane protein and developing models of helix packing interactions.

Keywords: Solid state NMR; Ion channel peptides; Cholesterol; Backbone secondary structure

Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52 by Wei Liu; Jeffrey Z. Fei; Toru Kawakami; Steven O. Smith (pp. 2971-2978).

The Ca²⁺-ATPase of cardiac muscle cells transports Ca²⁺ ions against a concentration gradient into the sarcoplasmic reticulum and is regulated by phospholamban, a 52-residue integral membrane protein. It is known that phospholamban inhibits the Ca²⁺ pump during muscle contraction and that inhibition is removed by phosphorylation of the protein during muscle relaxation. Phospholamban forms a pentameric complex with a central pore. The solid-state magic angle spinning (MAS) NMR measurements presented here address the structure of the phospholamban pentamer in the region of Gln22–Gln29. Rotational echo double resonance (REDOR) NMR measurements show that the side chain amide groups of Gln29 are in close proximity, consistent with a hydrogen-bonded network within the central pore.¹³C MAS NMR measurements are also presented on phospholamban that is 1-¹³C-labeled at Leu52, the last residue of the protein. pH titration of the C-terminal carboxyl group suggests that it forms a ring of negative charge on the lumenal side of the sarcoplasmic reticulum membrane. The structural constraints on the phospholamban pentamer described in this study are discussed in the context of a multifaceted mechanism for Ca²⁺ regulation that may involve phospholamban as both an inhibitor of the Ca²⁺ ATPase and as an ion channel.

Keywords: Abbreviations; DPC; dodecylphosphocholine; MAS; magic angle spinning; NMR; nuclear magnetic resonance; POPC; 1-palmitoyl, 2-oleoyl phosphocholine; POPS; 1-palmitoyl, 2-oleoyl phosphoserine; REDOR; rotational echo double resonance; SR; sarcoplasmic reticulumPhospholamban; Solid-state NMR; Transmembrane helix; Polarized infrared spectroscopy; Membrane reconstitution

Solid-State²H NMR spectroscopy of retinal proteins in aligned membranes by Michael F. Brown; Maarten P. Heyn; Constantin Job; Suhkmann Kim; Stephan Moltke; Koji Nakanishi; Alexander A. Nevzorov; Andrey V. Struts; Gilmar F.J. Salgado; Ingrid Wallat (pp. 2979-3000).

Solid-state²H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein.²H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C–C²H₃ groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state²H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all- trans light-adapted states, as well upon isomerization to 13- cis in the M state. The C13-methyl group at the “business end” of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11- cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the β-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by²H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state²H NMR is thus illuminating in terms of their different biological functions.

Keywords: Abbreviations; bR; bacteriorhodopsin; bR; all-; _t; bacteriorhodopsin with all-; trans; , 15-; anti; retinal chromophore; bR; _13-; _c; bacteriorhodopsin with 13-; cis; , 15-; syn; retinal chromophore; CD; circular dichroism; EFG; electric field gradient; GPCR; G protein-coupled receptor; M; bacteriorhodopsin M intermediate with 13-; cis; , 15-; anti; chromophore; MAS; magic-angle spinning; MD; molecular dynamics; meta I; metarhodopsin I; meta II; metarhodopsin II; NMR; nuclear magnetic resonance; PAS; principal axes system; PDB; Protein Data Bank; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSB; protonated Schiff base; PYP; photoactive yellow protein; RMSD; root mean square deviation; RQC; residual quadrupolar couplingBacteriorhodopsin; G protein-coupled receptor; Membrane; Proton pump; Solid-state NMR; Retinal; Rhodopsin; Vision

Structural analysis of pituitary adenylate cyclase-activating polypeptides bound to phospholipid membranes by magic angle spinning solid-state NMR by Nobuyasu Komi; Kayo Okawa; Yukihiro Tateishi; Masahiro Shirakawa; Toshimichi Fujiwara; Hideo Akutsu (pp. 3001-3011).

PACAP (pituitary adenylate cyclase-activating polypeptide) is a member of the VIP/secretin/glucagon family, which includes the ligands of class II G-protein coupled receptors. Since the recognition of PACAP by the receptor may involve the binding of PACAP to membranes, its membrane-bound structure should be important. We have carried out structural analysis of uniformly¹³C,¹⁵N labeled PACAP27 and its C-terminal truncated form PACAP(1–21)NH₂ (PACAP21) bound to membranes with high resolution solid-state NMR. Phosphatidylcholine bilayers and phosphatidylcholine/phosphatidylglycerol bilayers were used for PACAP27 and PACAP21, respectively. Most backbone signals were assigned for PACAP27 and PACAP21. TALOS analysis revealed that both peptides take on extended conformations on the membranes. Dilution of PACAP21 did not change the conformation of the major part. Selective polarization transfer experiment confirmed that PACAP27 is interacting with the membranes. It was concluded that the interaction of PACAP with the membrane surface causes their extended conformation. PACAP27 is reported to take an α-helical conformation in dodecylphosphocholine micelles and membrane-binding peptides usually take similar conformations in micelles and in membranes. Therefore, the property of PACAP27 changing its conformation in response to its environment is unique. Its conformational flexibility may be associated with its wide variety of functions.

Keywords: Abbreviations; CP; cross-polarization; DARR; dipolar assisted rotational resonance; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DPPG; 1,2-dipalmitoyl-; sn; -glycero-3-phosphatidylglycerol; DSS; 2,2-dimethylsilapenatane-5-sulfonic acid; LGCP; Lee–Goldburg cross polarization; PACAP; pituitary adenylate cyclase-activating polypeptide; RFDR; radio-frequency-driven recoupling; SPC-5; five-step super phase cycle; TPPM; two-pulse phase modulation; trNOE; transferred nuclear Overhauser effect; VIP; vasoactive intestinal polypeptidePituitary hormone; PACAP27; Membrane-bound peptide; Isotope-labeling; NMR signal assignment; Extended conformation

Proteorhodopsin: Characterisation of 2D crystals by electron microscopy and solid state NMR by Sarika Shastri; Janet Vonck; Nicole Pfleger; Winfried Haase; Werner Kuehlbrandt; Clemens Glaubitz (pp. 3012-3019).

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in γ-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by¹⁵N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee–Goldberg cross-polarisation has been used to probe protein backbone mobility.

Keywords: Abbreviations; (bR); bacteriorhodopsin; (CP); cross-polarisation; (CSA); chemical shift anisotropy; (CMC); critical micelle concentration; (DDM); n; -dodecyl β-; d; -maltoside; (DP); direct polarisation; (HETCOR); heteronuclear correlation; (OG); octyl-beta-; d; -glucopyranoside; (PR); proteorhodopsinSolid state NMR; 2D crystallography; Membrane protein; Reconstitution; Proteorhodopsin

¹³C and¹⁵N NMR evidence for peripheral intercalation of uniformly labeled fusogenic peptides incorporated in a biomimetic membrane by Prashant Agrawal; Suzanne Kiihne; Johan Hollander; Dieter Langosch; Huub de Groot (pp. 3020-3028).

Membrane fusion requires drastic and transient changes of bilayer curvature and here we have studied the interaction of three de novo designed synthetic hydrophobic peptides with a biomimetic three-lipid mixture by solid state NMR. An experimental approach is presented for screening of peptide–lipid interactions and their aggregation, and their embedding in a biomimetic membrane system using established proton-decoupled¹³C,¹⁵N and proton spin diffusion heteronuclear¹H−¹³C correlation NMR methods at high magnetic field. Experiments are presented for a set of de-novo designed fusion peptides in interaction with their lipid environment. The data provide additional support for the transmembrane model for the least fusogenic peptide, L16, while the peripheral intercalation model is preferred for the fusogenic peptides LV16 and LV16G8P9. This contributes to converging evidence that peripheral intercalation is both necessary and sufficient to trigger the fusion process for a lipid mixture close to a critical point for phase separation across the bilayer.

Keywords: Oriented bilayer; Transmembrane membrane fusogenic polypeptide; Membrane fusion; Solid state NMR

NMR crystallography: The effect of deuteration on high resolution¹³C solid state NMR spectra of a 7-TM protein by K. Varga; L. Aslimovska; I. Parrot; M.-T. Dauvergne; M. Haertlein; V.T. Forsyth; A. Watts (pp. 3029-3035).

The effect of deuteration on the¹³C linewidths of U-¹³C,¹⁵N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in²H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by <0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the Cδ, Cγ₁, and Cγ₂ Ile¹³C signals observed in the direct, 21–39 ppm, and indirect, 9–17 ppm, dimensions). The measured¹³C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies.¹H decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power¹H decoupling is required.

Keywords: Abbreviations; bR; bacteriorhodopsin; 7TM; 7-transmembrane; NMR; nuclear magnetic resonance; MAS; magic angle spinning; DGK; diaglycerol kinase; CP; cross polarisation; TPPM; two-pulse phase-modulated; Crh; catabolite repression histidine-containing phosphocarrier protein; DARR; dipolar assisted rotational resonance; DSS; 2,2-dimethyl-2-silapentane-5-sulfonate; S/N; single-to-noise ratio; CT; contact time; HETCOR; heteronuclear correlationMMR crystallography; Membrane protein structure; Bacteriorhodopsin

MAS solid-state NMR studies on the multidrug transporter EmrE by Vipin Agarwal; Uwe Fink; Shimon Schuldiner; Bernd Reif (pp. 3036-3043).

We study the uniformly¹³C,¹⁵N isotopically enriched Escherichia coli multidrug resistance transporter EmrE using MAS solid-state NMR. Solid-state NMR can provide complementary structural information as the method allows studying membrane proteins in their native environment as no detergent is required for reconstitution. We compare the spectra obtained from wildtype EmrE to those obtained from the mutant EmrE-E14C. To resolve the critical amino acid E14, glutamic/aspartic acid selective experiments are carried out. These experiments allow to assign the chemical shift of the carboxylic carbon of E14. In addition, spectra are analyzed which are obtained in the presence and absence of the ligand TPP+.

Keywords: MAS solid-state NMR; Multidrug resistance transporter; Membrane protein

The measurement of immersion depth and topology of membrane proteins by solution state NMR by R. Scott Prosser; Ferenc Evanics; Julianne L. Kitevski; Sagar Patel (pp. 3044-3051).

An important component of the study of membrane proteins involves the determination of details associated with protein topology — for example, the location of transmembrane residues, specifics of immersion depth, orientation of the protein in the membrane, and extent of solvent exposure for each residue. Solution state NMR is well suited to the determination of immersion depth with the use of paramagnetic additives designed to give rise to depth-specific relaxation effects or chemical shift perturbations. Such additives include spin labels designed to be “anchored” within a given region of the membrane or small freely diffusing paramagnetic species, whose partitioning properties across the water membrane interface create a gradient of paramagnetic effects which correlate with depth. This review highlights the use of oxygen and other small paramagnetic additives in studies of immersion depth and topology of membrane proteins in lipid bilayers and micelles.

Keywords: Solution NMR; Membrane protein; Immersion depth; Oxygen; Paramagnetic effect

Combined NMR and EPR spectroscopy to determine structures of viral fusion domains in membranes by Lukas K. Tamm; Alex L. Lai; Yinling Li (pp. 3052-3060).

Methods are described to determine the structures of viral membrane fusion domains in detergent micelles by NMR and in lipid bilayers by site-directed spin labeling and EPR spectroscopy. Since in favorable cases, the lower-resolution spin label data obtained in lipid bilayers fully support the higher-resolution structures obtained by solution NMR, it is possible to graft the NMR structural coordinates into membranes using the EPR-derived distance restraints to the lipid bilayer. Electron paramagnetic dynamics and distance measurements in bilayers support conclusions drawn from NMR in detergent micelles. When these methods are applied to a structure determination of the influenza virus fusion domain and four point mutations with different functional phenotypes, it is evident that a fixed-angle boomerang structure with a glycine edge on the outside of the N-terminal arm is both necessary and sufficient to support membrane fusion. The human immunodeficiency virus fusion domain forms a straight helix with a flexible C-terminus. While EPR data for this fusion domain are not yet available, it is tentatively speculated that, because of its higher hydrophobicity, a critically tilted insertion may occur even in the absence of a kinked boomerang structure in this case.

Keywords: Solution spectroscopy; EPR spectroscopy; Membrane fusion domain; Influenza virus; Human immunodeficiency virus

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy by Sanjeewa G. Rupasinghe; Hui Duan; Heather L. Frericks Schmidt; Deborah A. Berthold; Chad M. Rienstra; Mary A. Schuler (pp. 3061-3070).

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, Escherichia coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of¹³C- and¹⁵N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis¹³C,¹⁵N-labeled His₄CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated His₄CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.

Keywords: Cytochrome P450 monooxygenases (P450s); Heterologous expression; Membrane protein; Solid-state NMR analysis

Structural characterization of the pore forming protein TatA_d of the twin-arginine translocase in membranes by solid-state¹⁵N-NMR by Sonja D. Müller; Anna A. De Angelis; Torsten H. Walther; Stephan L. Grage; Christian Lange; Stanley J. Opella; Anne S. Ulrich (pp. 3071-3079).

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2–45 of TatA_d from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatA_d was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in¹⁵N chemical shift /¹⁵N–¹H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14–16 residue long N-terminal membrane-spanning helix which is tilted by 17° with respect to the membrane normal. In addition, comparison of uniformly and selectively¹⁵N-labeled TatA_2–45 samples allowed determination of the helix polarity angle.

Keywords: Abbreviations; Tat; twin-arginine translocation; B. subtilis; Bacillus subtilis; E. coli; Escherichia coli; CD; circular dichroism; OCD; oriented circular dichroism; NLS; N-lauroylsarcosine; NMR; nuclear magnetic resonance; MALDI-TOF; matrix assisted laser desorption ionisation-time of flight; SDS-PAGE; sodium dodecyl sulphate-polyacrylamide gel electrophoresis; IPTG; isopropyl-β-D-thiogalactopyranosid; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)]; DHPC; 1,2-dihexanoyl-; sn; -glycero-3-phosphocholine, 6-O-PC: 1,2-; O; -dihexyl-; sn; -glycero-3-phosphocholine; TMS; transmembrane segment; APH; amphiphilic helix; TatA; _2–45; protein fragment containing the transmembrane and central segments; FID; free induction decay; HSQC; heteronuclear single-quantum coherence; PISA wheel; polarity index slant angle wheel; PISEMA; polarization inversion spin exchange at the magic angle; CP-MOIST; cross-polarization with mismatch optimized IS transferTwin-arginine translocation; TatA; _d; Bacillus subtilis; Membrane; PISEMA solid-state NMR; PISA wheel

S100A13–lipid interactions—role in the non-classical release of the acidic fibroblast growth factor by Karuppanan Muthusamy Kathir; Khalil Ibrahim; Dakshinamurthy Rajalingam; Igor Prudovsky; Chin Yu; Thallapuranam Krishnaswamy Suresh Kumar (pp. 3080-3089).

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles ofl-phosphatidyl serine (pS). In comparison, Ca²⁺-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca²⁺ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.

Keywords: Acidic fibroblast growth factor; Calcium binding; Lipid binding; Non-classical secretion; Multi-protein complex; Hydrophobic

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR by Izuru Kawamura; Masato Ohmine; Junko Tanabe; Satoru Tuzi; Hazime Saitô; Akira Naito (pp. 3090-3097).

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved¹³C and¹⁵N signals of [1-¹³C]Tyr-, [¹⁵N]Pro- and [1-¹³C]Val-, [¹⁵N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-¹³C]Val-, [¹⁵N]Pro signals to the specific residues in bR. The previous assignments of [1-¹³C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent¹⁵N chemical shifts of Pro bonded to Val in the presence of¹³C–¹⁵N correlation, although no assignment of peak is feasible for¹³C nuclei not bonded to Pro.¹³C or¹⁵N spin–lattice relaxation times ( T₁), spin–spin relaxation times under the condition of CP-MAS ( T₂), and cross relaxation times ( T_CH and T_NH) for¹³C and¹⁵N nuclei and carbon or nitrogen-resolved,¹H spin–lattice relaxation times in the rotating flame (¹H T_1ρ) for the assigned signals were measured in [1-¹³C]Val-, [¹⁵N]Pro-bR. It turned out that V69–P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from¹³C and¹⁵N conformation-dependent chemical shifts and T₁, T₂,¹H T_1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.

Keywords: Abbreviations; bR; bacteriorhodopsin; bO; bacterio-opsin; PM; purple membrane; CP-MAS; Cross polarization-magic angle spinning; REDOR; Rotational echo double resonanceBacteriorhodopsin; Bacterio-opsin; Relaxation time; Rotational echo double resonance; Solid state NMR

Solution NMR of membrane proteins in bilayer mimics: Small is beautiful, but sometimes bigger is better by Sébastien F. Poget; Mark E. Girvin (pp. 3098-3106).

Considerable progress has been made recently on solution NMR studies of multi-transmembrane helix membrane protein systems of increasing size. Careful correlation of structure with function has validated the physiological relevance of these studies in detergent micelles. However, larger micelle and bicelle systems are sometimes required to stabilize the active forms of dynamic membrane proteins, such as the bacterial small multidrug resistance transporters. Even in these systems with aggregate molecular weights well over 100 kDa, solution NMR structural studies are feasible—but challenging.

Keywords: Micelle; Bicelle; Structure; Smr; Multidrug resistance

Dual transformation of homonuclear solid-state NMR spectra—an option to decrease measuring time by Christoph Kaiser; Jakob J. Lopez; Wolfgang Bermel; Clemens Glaubitz (pp. 3107-3115).

Long measurement times due to low sensitivity are a prime concern in solid-state NMR and limit the application of multidimensional experiments severely. One possibility to address this problem could be post-experimental suppression of noise and a reduction of the number of increments needed for higher dimensional data sets. This can be achieved by a hybrid approach based on the combination of separately Fourier transformed and covariance processed datasets. The method is applied to synthetic sets as well as to experimental two-dimensional homonuclear solid-state NMR spectra of peptide samples. It is demonstrated that a reduction in experiment time by a factor of 4 can be achieved for the case of a¹³C–¹³C correlation spectrum on the nonapeptide bradykinin.

Keywords: Abbreviations; ssNMR; solid state nuclear magnetic resonance; PDSD; proton driven spin diffusion; DARR; dipolar assisted rotational resonance; DTD; dual transformation denoising; SPINAL; small phase incremental alteration; MLF; methionine–leucine–phenylalanine tripeptide; S/N; signal to noise ratio; FFT; fast Fourier transformDenoising; Fast NMR; Solid state NMR; MAS; Bradykinin; Covariance

NMR structural studies of the antibiotic lipopeptide daptomycin in DHPC micelles by Walter R.P. Scott; Seung-Bin Baek; David Jung; Robert E.W. Hancock; Suzana K. Straus (pp. 3116-3126).

Daptomycin is a cyclic anionic lipopeptide that exerts its rapid bactericidal effect by perturbing the bacterial cell membrane, a mode of action different from most other currently commercially available antibiotics (except e.g. polymyxin and gramicidin). Recent work has shown that daptomycin requires calcium in the form of Ca²⁺ to form a micellar structure in solution and to bind to bacterial model membranes. This evidence sheds light on the initial steps in the mechanism of action of this novel antibiotic. To understand how daptomycin goes on to perturb bacterial membranes, its three-dimensional structure has been determined in the presence of 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC) micelles. NMR spectra of daptomycin in DHPC were obtained under two conditions, namely in the presence of Ca²⁺ as used by Jung et al. [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949–57] to solve the calcium-conjugated structure of daptomycin in solution and in a phosphate buffer as used by Rotondi and Gierasch [K.S. Rotondi, L.M. Gierasch, A well-defined amphipathic conformation for the calcium-free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution, Biopolymers 80 (2005) 374–85] to solve the structure of apo-daptomycin. The structures were calculated using molecular dynamics time-averaged refinement. The different sample conditions used to obtain the NMR spectra are discussed in light of fluorescence data, lipid flip-flop and calcein release assays in PC liposomes, in the presence and absence of Ca²⁺ [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium–dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949–57]. The implications of these results for the membrane perturbation mechanism of daptomycin are discussed.

Keywords: Daptomycin; Lipopeptide; Solution state NMR; NOEs; Structure calculation; Mechanism of action

NMR and mutagenesis of human copper transporter 1 (hCtr1) show that Cys-189 is required for correct folding and dimerization by Sangwon Lee; Stephen B. Howell; Stanley J. Opella (pp. 3127-3134).

The human high-affinity copper transporter (hCtr1) is a membrane protein that is predicted to have three transmembrane helices and two methionine-rich metal binding motifs. As an oligomeric polytopic membrane protein, hCtr1 is a challenging system for experimental structure determination. The results of an initial application of solution-state NMR methods to a truncated construct containing residues 45–190 in micelles and site-directed mutagenesis of the two cysteine residues demonstrate that Cys-189 but not Cys-161 is essential for both dimer formation and proper folding of the protein.

Keywords: Oligomerization; Copper transport; hCtr1; NMR; Disulfide bond

Cholesterol and Clioquinol modulation of Aβ(1–42) interaction with phospholipid bilayers and metals by Tong-Lay Lau; John D. Gehman; John D. Wade; Colin L. Masters; Kevin J. Barnham; Frances Separovic (pp. 3135-3144).

The β-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-β peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, Aβ(1–42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state³¹P and²H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine–phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in β-sheet, were observed using specifically¹³C-labelled Aβ(1–42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely¹⁵N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric β-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.

Keywords: Amyloid Aβ; Peptide–lipid interaction; Phospholipid membrane; Solid-state NMR; Structure; Cholesterol; Aligned lipid bilayer

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein by Hazime Saitô; Akira Naito (pp. 3145-3161).

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.

Keywords: Abbreviations; bR; bacteriorhodopsin; bO; bacterioopsin; Bchl; bacteriochlorophylls; CP-MAS; cross polarization-magic angle spinning; 2D; two dimensional; 3D; three dimensional; DARR; dipolar-assisted rotational resonance; DD-MAS; a single-pulse; ¹³; C dipolar decoupled-magic angle spinning; DGK; diacylglycerol kinase; DM; N; -dodecylmaltoside; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl- sn-glycero-3-phosphocholine; DSS; 2,2-dimethylsilapentane-5-sulfonic acid; EC; extracellular; GPCRs; G-protein-coupled receptors; HSQC; heteronuclear single quantum coherence; HETCOR; heteronuclear correlation; HFIP; hexafluoroisopropanol; IP; ₃; d; -; myo; -inositol 1,4,5-trisphosphate; LH2; light-harvesting complex; MAS; magic angle spinning; MOVS; magnetically oriented vesicle system; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; OTG; octyl ß-glucoside; p; HtrII; pharaonis; cognate transducer; PM; purple membrane; PISA; polarity index slant angle; PISEMA; polarization inversion spin exchange at the magic angle; PH; pleckstrin homology; PIP; ₂; phosphatidylinositol 4,5-bisphosphate; PLC; phospholipase C; POPC; 1-palmitoyl-2-oleyl-; sn; -glycero-3-phosphocholine; pR; phoborhodopsin; p; pR; pharaonis; phoborhodopsin; RC; reaction center; REDOR; rotational echo double resonance; RFDR; radiofrequency-driven recoupling; Rho; rhodopsin; SDS; sodium dodecyl sulfate; SH3; Src homology 3; sR I; sensory rhodopsin I; sR II; sensory rhodopsin II; TET; trifluoroethylthio; TM; transmembrane; TMS; tetramethylsilane; TX-100; Triton X-100; TN-101; Triton N-101; TROSY; transverse relaxation-optimized NMR spectroscopyNMR; Membrane proteins; Fully hydrated; ¹³; C NMR; Surface structure; Dynamics; Conformation

Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel by Conggang Li; Huajun Qin; Fei Philip Gao; Timothy A. Cross (pp. 3162-3170).

Membrane protein function within the membrane interstices is achieved by mechanisms that are not typically available to water-soluble proteins. The whole balance of molecular interactions that stabilize three-dimensional structure in the membrane environment is different from that in an aqueous environment. As a result interhelical interactions are often dominated by non-specific van der Waals interactions permitting dynamics and conformational heterogeneity in these interfaces. Here, solid-state NMR data of the transmembrane domain of the M2 protein from influenza A virus are used to exemplify such conformational plasticity in a tetrameric helical bundle. Such data lead to very high resolution structural restraints that can identify both subtle and substantial structural differences associated with various states of the protein. Spectra from samples using two different preparation protocols, samples prepared in the presence and absence of amantadine, and spectra as a function of pH are used to illustrate conformational plasticity.

Keywords: M2 channel; Influenza A virus; Conformational plasticity; PISEMA; Solid-state NMR; Membrane protein

Characterization of the myristoyl lipid modification of membrane-bound GCAP-2 by²H solid-state NMR spectroscopy by Alexander Vogel; Thomas Schröder; Christian Lange; Daniel Huster (pp. 3171-3181).

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca²⁺ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca²⁺-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and²H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the²H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of −0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain–lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases.

Keywords: Abbreviations; cGMP; guanosine 3′:5′-monophosphate; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPC-; d; ₅₄; 1,2-myristoyl-; d; ₅₄; -; sn; -glycero-3-phosphocholine; GCAP-2; guanylyl cyclase-activating protein-2; NCS; neuronal Ca; ²⁺; -sensor proteins; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPC-; d; ₃₁; 1-palmitoyl-; d; ₃₁; -2-oleoyl-; sn; -glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; POPE-; d; ₃₁; 1-palmitoyl-; d; ₃₁; -2-oleoyl-; sn; -glycero-3-phosphoethanolamine; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylserine; POPS-; d; ₃₁; 1-palmitoyl-; d; ₃₁; -2-oleoyl-; sn; -glycero-3-phosphatidylserine; ROS-GC; retinal guanylate cyclaseGuanylate cyclase-activating protein; Neuronal calcium sensor; Membrane binding; Myristoylation; Lipid modification; ²; H solid-state NMR; Order parameter

Structural biology of membrane-acting peptides: Conformational plasticity of anticoccidial peptide PW2 probed by solution NMR by C. Cruzeiro-Silva; F. Gomes-Neto; L.W. Tinoco; E.M. Cilli; P.V.R. Barros; P.A. Lapido-Loureiro; P.M. Bisch; F.C.L. Almeida; A.P. Valente (pp. 3182-3192).

The bottleneck for the complete understanding of the structure–function relationship of flexible membrane-acting peptides is its dynamics. At the same time, not only the structure but also the dynamics are the key points for their mechanism of action. Our model is PW2, a TRP-rich, cationic peptide selected from phage display libraries that shows anticoccidial activity against Eimeria acervulina. In this manuscript we used a combination of several NMR techniques to tackle these difficulties. The structural features of the membrane-acting peptide PW2 was studied in several membrane mimetic environments: we compared the structural features of PW2 in SDS and DPC micelles, that were reported earlier, with the structure properties in different lipid vesicles and the peptide free in water. We were able to unify the structural information obtained in each of these systems. The structural constraints of the peptide free in water were fundamental for the understanding of plasticity necessary for the membrane interaction. Our data suggested that the WWR sequence is the region responsible for anchoring the peptide to the interfaces, and that this same region displays some degree of conformational order in solution. For PW2, we found that affinity is related to the aromatic region, by anchoring the peptide to the membrane, and specificity is related to the N- and C-termini, which are able to accommodate in the membrane due to its plasticity.

Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: Spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments by Ligang Zhong; Vladimir V. Bamm; Mumdooh A.M. Ahmed; George Harauz; Vladimir Ladizhansky (pp. 3193-3205).

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein–lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.

Keywords: Abbreviations; CARA; computer-aided resonance assignment; CP; cross-polarization; CPMAS; cross-polarization magic angle spinning; CSA; chemical shift anisotropy; CSI; chemical shift index; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)]; ddH; ₂; O; distilled, deionised water; EDTA; ethylenediamine tetraacetic acid; EPR; electron paramagnetic resonance; FID; free induction decay; GARP; globally optimised alternating phase rectangular pulses; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HSQC; heteronuclear single quantum coherence; INEPT; insensitive nuclear enhancement of polarization transfer; LUV; large unilamellar vesicle; MAS; magic angle spinning; MBP; myelin basic protein; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; ppm; parts per million; rmMBP; recombinant murine MBP; rpm; revolutions per minute; SDS; sodium dodecyl sulphate; SDSL; site-directed spin labelling; ssNMR; solid-state NMR; TFE-d; ₂; deuterated 2,2,2-trifluoroethanol (CF; ₃; -CD; ₂; -OH); TOBSY; total through-bond correlation spectroscopy; TPPI; time-proportional phase incrementation; TPPM; two-pulse phase modulation; Tris–HCl; tris; (hydroxymethyl)aminomethane, pH adjusted with HCl; WALTZ; wideband, alternating phase, low-power technique for zero-residual splittingMyelin basic protein (MBP); Solid-state NMR; Magic angle spinning; J-couplings; INEPT; TOBSY; Multidimensional NMR

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR by Yanqiu Wu; Steve C.C. Shih; Natalie K. Goto (pp. 3206-3215).

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.

Keywords: Abbreviations; TM; transmembrane; GpA; glycophorin A; MCP; major coat protein; SDS; sodium dodecyl sulfate; β-OG; β-octyl glucoside; TFA; trifluoroacetic acid; HPLC; high performance liquid chromatography; BCA; bicinchoninic acid; BSA; bovine serum albumin; MTSL; 1-oxyl-2,2,5,5-tetramethyl-Δ; ³; -pyrrolin-3-yl methyl methanethiosulfonate; CD; circular dichroism; HSQC; heteronuclear single quantum coherence; TOCSY; total correlation spectroscopy; NOESY; nuclear Overhauser enhancement spectroscopy; PRE; paramagnetic relaxation enhancement; AIR; ambiguous interaction restraints; C; ₈; E; ₅; n; -octyl pentaoxyethylene glycol monoetherSolution NMR; Transmembrane peptide; Helix–helix interaction; Ff bacteriophage major coat protein; Paramagnetic relaxation enhancement

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes by Radhakrishnan Mahalakshmi; Carla M. Franzin; Jungyuen Choi; Francesca M. Marassi (pp. 3216-3224).

The β-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D₂O for days and is assigned to the transmembrane β-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D₂O are assigned to the extracellular and periplasmic loops. The two-dimensional¹H/¹⁵N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5° tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to α-helical membrane proteins.

Keywords: OmpX; Beta barrel; Outer membrane protein; Bilayer; Bicelle; Solid state NMR

Chemical shift assignment and structural plasticity of a HIV fusion peptide derivative in dodecylphosphocholine micelles by Charles M. Gabrys; David P. Weliky (pp. 3225-3234).

A “HFPK3” peptide containing the 23 residues of the human immunodeficiency virus (HIV) fusion peptide (HFP) plus three non-native C-terminal lysines was studied in dodecylphosphocholine (DPC) micelles with 2D¹H NMR spectroscopy. The HFP is at the N-terminus of the gp41 fusion protein and plays an important role in fusing viral and target cell membranes which is a critical step in viral infection. Unlike HFP, HFPK3 is monomeric in detergent-free buffered aqueous solution which may be a useful property for functional and structural studies. H^α chemical shifts indicated that DPC-associated HFPK3 was predominantly helical from I4 to L12. In addition to the highest-intensity crosspeaks used for the first chemical shift assignment (denoted I), there were additional crosspeaks whose intensities were ∼10% of those used for assignment I. A second assignment (II) for residues G5 to L12 as well as a few other residues was derived from these lower-intensity crosspeaks. Relative to the I shifts, the II shifts were different by 0.01–0.23 ppm with the largest differences observed for H^N. Comparison of the shifts of DPC-associated HFPK3 with those of detergent-associated HFP and HFP derivatives provided information about peptide structures and locations in micelles.

Keywords: Abbreviations; COSY; correlation spectroscopy; DPC; dodecylphosphocholine; DQ-COSY; double-quantum filtered correlation spectroscopy; DSS; 3-(trimethylsilyl)-1-propanesulfonic acid, sodium salt; HEPES; N; -(2-hydroxyethyl)piperazine-; N; ′-2-ethanesulfonic acid; HFP; HIV fusion peptide; HFPK3; HIV fusion peptide plus three lysines; HFPK3W; HIV fusion peptide plus three lysines and a tryptophan; HIV; human immunodeficiency virus; NMR; nuclear magnetic resonance; NOESY; nuclear Overhauser effect spectroscopy; SDS; sodium dodecyl sulfate; TOCSY; total correlation spectroscopy; WET; water suppression enhanced through; T; ₁; effects; 2D; two-dimensionalDPC; Fusion peptide; HIV; NMR; Structural heterogeneity; Cryoprobe; SDS

The cytochromes P450 and b₅ and their reductases—Promising targets for structural studies by advanced solid-state NMR spectroscopy by Ulrich H.N. Dürr; Lucy Waskell; Ayyalusamy Ramamoorthy (pp. 3235-3259).

Members of the cytochrome P450 (cyt P450) superfamily of enzymes oxidize a wide array of endogenous and xenobiotic substances to prepare them for excretion. Most of the drugs in use today are metabolized in part by a small set of human cyt P450 isozymes. Consequently, cyt P450s have for a long time received a lot of attention in biochemical and pharmacological research. Cytochrome P450 receives electrons from cytochrome P450 reductase and in selected cases from cytochrome b₅ (cyt b₅). Numerous structural studies of cyt P450s, cyt b₅, and their reductases have given considerable insight into fundamental structure–function relationships. However, structural studies so far have had to rely on truncated variants of the enzymes to make conventional X-ray crystallographic and solution-state NMR techniques applicable. In spite of significant efforts it has not yet been possible to crystallize any of these proteins in their full-length membrane bound forms. The truncated parts of the enzymes are assumed to be α-helical membrane anchors that are essential for some key properties of cyt P450s. In the present contribution we set out with a basic overview on the current status of functional and structural studies. Our main aim is to demonstrate how advanced modern solid-state NMR spectroscopic techniques will be able to make substantial progress in cyt P450 research. Solid-state NMR spectroscopy has sufficiently matured over the last decade to be fully applicable to any membrane protein system. Recent years have seen a remarkable increase in studies on membrane protein structure using a host of solid-state NMR techniques. Solid-state NMR is the only technique available today for structural studies on full-length cyt P450 and full-length cyt b₅. We aim to give a detailed account of modern techniques as applicable to cyt P450 and cyt b₅, to show what has already been possible and what seems to be viable in the very near future.

Keywords: Abbreviations; b5R; cytochrome; b; ₅; reductase; CP; cross-polarization; CPR; cytochrome P450 reductase; CSA; chemical shift anisotropy; CYP; cytochrome P450; cyt; b; ₅; cytochrome; b; ₅; cyt P450; cytochrome P450; DHPC; dihexanoylphosphatidylcholine; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidylglycerol; DPPC; dipalmitoylphosphatidylcholine; ER; endoplasmic reticulum; FAD; flavin adenine dinucleotide; FMN; flavin mononucleotide; HIMSELF; heteronuclear isotropic mixing by spin exchange at the magic angle; MAS; magic angle spinning; MLV; multilamellar vesicles; NADH; nicotinamide adenine dinucleotide; NADPH; nicotinamide adenine dinucleotide phosphate; NMR; nuclear magnetic resonance; PISEMA; polarization inversion by spin exchange at the magic angle; RINEPT; refocused insensitive nuclei enhancement by polarization transfer; TM; transmembraneMembrane protein; Solid-state NMR; Cytochrome P450; Cytochrome; b; ₅

Local and global structure of the monomeric subunit of the potassium channel KcsA probed by NMR by Jordan H. Chill; John M. Louis; Frank Delaglio; Ad Bax (pp. 3260-3270).

KcsA is a homotetrameric 68-kDa membrane-associated potassium channel which selectively gates the flux of potassium ions across the membrane. The channel is known to undergo a pH-dependent open-to-closed transition. Here we describe an NMR study of the monomeric subunit of the channel (KcsA^M), solubilized in SDS micelles. Chemical shift, solvent exchange, backbone¹⁵N relaxation and residual dipolar coupling (RDC) data show the TM1 helix to remain intact, but the TM2 helix contains a distinct kink, which is subject to concentration-independent but pH-dependent conformational exchange on a microsecond time scale. The kink region, centered at G99, was previously implicated in the gating of the tetrameric KcsA channel. An RDC-based model of KcsA^M at acidic pH orients TM1 and the two helical segments of the kinked TM2 in a configuration reminiscent of the open conformation of the channel. Thus, the transition between states appears to be an inherent capability of the monomer, with the tetrameric assembly exerting a modulatory effect upon the transition which gives the channel its physiological gating profile.

Keywords: Membrane proteins; Potassium channel; NMR; RDC; Solvent exchange

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample by Guangshun Wang (pp. 3271-3281).

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain H^N protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide–lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the εH^N protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selectived-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein–lipid NOE cross peaks are discussed.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; D8PG; dioctanoyl phosphatidylglycerol; DPC; dodecylphosphocholine; DQF-COSY; double-quantum filtered correlation spectroscopy; DSS; 2,2-dimethyl-silapentane-5-sulfonate sodium salt; HSQC; heteronuclear single-quantum coherence spectroscopy; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser enhancement spectroscopy; PGs; phosphatidylglycerols; rmsd; root mean square deviation; SDS; sodium dodecylsulfate; TOCSY; total correlation spectroscopy; TOCSY-trim; a technique for identification of the key membrane-binding region of a peptide by trimming off nonessential portions based on strong TOCSY relaysAntimicrobial peptide; D8PG; Intermolecular NOE; Membrane peptide; NMR; Peptide–lipid interaction; Structural biology

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide by Anirban Bhunia; Prerna N. Domadia; Surajit Bhattacharjya (pp. 3282-3291).

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant ( K_a) of 2.85×10⁶ M−¹ and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (Δ G⁰), −8.8 kcal mol−¹, obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.

Keywords: LPS; Melittin; NMR; Transferred nuclear Overhauser effect; Saturation transfer difference; STD; REES

Sections

Personal tools

BBA - Biomembranes (v.1768, #12)

Sections

Personal tools

Local resources

BBA - Biomembranes (v.1768, #12)