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BBA - Biomembranes (v.1798, #2)

Editorial Board (pp. i).

Special issue on “Membrane Protein Dynamics: Correlating Structure to Function” by Gianluigi Veglia; Ayyalusamy Ramamoorthy (pp. 65-67).

Fast-time scale dynamics of outer membrane protein A by extended model-free analysis of NMR relaxation data by Binyong Liang; Ashish Arora; Lukas K. Tamm (pp. 68-76).

In order to better understand the dynamics of an integral membrane protein, backbone amide¹⁵N NMR dynamics measurements of the β-barrel membrane protein OmpA have been performed at three magnetic fields. A total of nine relaxation data sets were globally analyzed using an extended model-free formalism. The diffusion tensor was found to be prolate axially symmetric with an axial ratio of 5.75, indicating a possible rotation of the protein within the micelle. The generalized order parameters gradually decreased from the mid-plane towards the two ends of the barrel, counteracting the dynamic gradient of the lipids in a matching bilayer, and were dramatically reduced in the extracellular loops. Large-scale internal motions on the ns time scale indicate that entire loops most likely undergo concerted (“sea anemone”-like) motions emanating from their anchoring points on the barrel. The case of OmpA in DPC micelles also illustrates inherent limitations of analyzing the data with even the most sophisticated current models of the model-free formalism. It is likely that conformational exchange processes on the ms–μs also play a role in describing the motions of some residues, but their analysis did not produce unique results that could be independently verified.

Keywords: Membrane protein; Dynamics; NMR; β-barrel; Model-free

Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments by Nathaniel J. Traaseth; Gianluigi Veglia (pp. 77-81).

Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca²⁺ ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca²⁺ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the μs to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The¹⁵N backbone and¹³C_δ1 Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1–16), the loop (17–22), and domain Ib (23–30) of PLN undergo μs-ms dynamics ( k_ex=6100±800 s-¹ at 17 °C). We measured k_ex at additional temperatures, which allowed for a calculation of activation energy equal to ∼5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners.

Keywords: Abbreviations; Phospholamban; PLN; sarco(endo)plasmic reticulum Ca-ATPase; SERCA; Carr–Purcell–Meiboom–Gill; CPMGPhospholamban; NMR; Protein Dynamics; Relaxation Dispersion; Membrane Protein; Ca; ²⁺; -ATPase; SERCA; Solution NMR

Interactions between the human sweet-sensing T1R2–T1R3 receptor and sweeteners detected by saturation transfer difference NMR spectroscopy by Fariba M. Assadi-Porter; Marco Tonelli; Emeline L. Maillet; John L. Markley; Marianna Max (pp. 82-86).

The sweet receptor is a member of the G-protein coupled receptor family C that detects a wide variety of chemically and structurally diverse sweet-tasting molecules. We recently used saturation transfer difference spectroscopy (STD) to monitor the direct binding of a set of sweet agonists and antagonists to the human taste receptor in membranes prepared from human embryonic kidney (HEK293) cells transfected with and expressing the sweet receptor [F.M. Assadi-Porter, M. Tonelli, E. Maillet, K. Hallenga, O. Benard, M. Max, J.L. Markley, J. Am. Chem. Soc. 130 (2008) 7212–7213]. Here we review this work and related studies, discuss the procedures involved, and expand on their potential for identifying specific binding interactions of ligands to the membrane spanning and extracellular regions of the full heterodimeric sweet taste receptor. Whereas activity assays are unable to distinguish mutations that alter ligand-binding sites from those that alter signal transduction downstream of the binding site, STD NMR now allows us to make this distinction.

Keywords: Ligand binding; Sweet receptor; G-protein coupled receptor (GPCR); Saturation transfer difference (STD); Nuclear magnetic resonance (NMR) spectroscopy; Brazzein

NMR dynamics and antibody recognition of the meningococcal lipidated outer membrane protein LP2086 in micellar solution by Alessandro Mascioni; Franklin J. Moy; Lisa K. McNeil; Ellen Murphy; Breagh E. Bentley; Rosaria Camarda; Deborah A. Dilts; Pamela S. Fink; Viktoria Gusarova; Susan K. Hoiseth; Karl Malakian; Terri Mininni; Elena Novikova; Shuo Lin; Scott Sigethy; Gary W. Zlotnick; Désirée H.H. Tsao (pp. 87-93).

Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.

Keywords: Abbreviations; fHBP; factor H binging protein; PorA; porin A; PorB; porin B; rLP2086; recombinant lipidated P2086; rP2086; recombinant non-lipidated P2086; rLP2086-B01; recombinant lipidated LP2086 variant B01; LOS; lipo-oligosaccharides; LPS; lipopolysaccharides; mAb; monoclonal antibodyOuter membrane protein; Detergent micelle; Lipoprotein; Neisseria meningitidis; LP2086; Factor H binding protein; NMR spectroscopy; Structure; Dynamics; Membrane topology

Correlating structure, dynamics, and function in transmembrane segment VII of the Na⁺/H⁺ exchanger isoform 1 by Tyler Reddy; Xiuju Li; Larry Fliegel; Brian D. Sykes; Jan K. Rainey ⁎ (pp. 94-104).

We place¹⁵N nuclear magnetic resonance relaxation analysis and functional mutagenesis studies in the context of our previous structural and mutagenesis work to correlate structure, dynamics and function for the seventh transmembrane segment of the human Na⁺/H⁺ exchanger isoform 1. Although G261–S263 was previously identified as an interruption point in the helical structure of this isolated transmembrane peptide in dodecylphosphocholine micelles, and rapid conformational exchange was implicated in the NOE measurements, the six¹⁵N labelled residues examined in this study all have similar dynamics on the ps–ns time scale. A mathematical model incorporating chemical exchange is the best fit for residues G261, L264, and A268. This implies that a segment of residues from G261 to A268 samples different conformations on the μs–ms time scale. Chemical exchange on an intermediate time scale is consistent with an alternating-access cycle where E262 is bent away from the cytosol during proton translocation by the exchanger. The functional importance of chemical exchange at G261–A268 is corroborated by the abrogated activity of the full-length exchanger with the bulky and restricting Ile substitutions F260I, G261I, E262I, S263I, and A268I.

Keywords: Abbreviations; AIC; Akaike's information criteria; CSA; chemical shift anisotropy; DPC(-; d; ₃₈; ); (deuterated) dodecylphosphocholine; NhaA; prokaryotic Na; ⁺; /H; ⁺; antiporter; NHE1; Na; ⁺; /H; ⁺; exchanger isoform 1; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; TM; transmembraneNMR; ¹⁵; N relaxation; Model-Free analysis; Reduced spectral density mapping; DPC; Peptide

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy by Luciano Neves de Medeiros; Renata Angeli; Carolina G. Sarzedas; Eliana Barreto-Bergter; Ana Paula Valente; Eleonora Kurtenbach; Fabio C.L. Almeida (pp. 105-113).

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2¹⁵N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36–Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs–ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.

Keywords: Abbreviations; CMH; monohexosylceramide; CSP; chemical shift perturbation; Dm; AMP; Dahlia merckii; antimicrobial peptide; DPC; dodecylphosphocholine; Het-NOE; Heteronuclear; ¹⁵; N Nuclear Overhauser Enhancement; -M(IP); ₂; C); mannosyldiinositolphosphoryl-ceramide; pepLoop1; Gly12 to Ser19; Psd; Pisum sativum; defensin; Rs; AFP1; Raphanus sativus; antifungal peptide; PC; l; -α-phosphatidylcholine; PRE; paramagnetic relaxation enhancement; R1; heteronuclear longitudinal relaxation time; R2; heteronuclear transverse relaxation timePlant defensin; Membrane; Membrane protein; NMR; Dynamic; Lipari–Szabo; Antimicrobial; Fungus; Protein recognition; Membrane recognition; Transient interaction; Psd1; Antifungal

Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance¹³C NMR spectroscopy by Guangshun Wang (pp. 114-121).

Worldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance¹³C NMR spectroscopy that provides complementary information to 2D¹H NMR. The correlation of¹³Cα secondary shifts with both 3D structure and heteronuclear¹⁵N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D¹H–¹³Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles.¹H–¹³Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide–membrane interactions from different angles.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; AMPs; antimicrobial peptides; APD2; the antimicrobial peptide database version 2; 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; LLAA; LL-37-derived aurein 1.2 analog; MIC; minimal inhibitory concentration; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser enhancement spectroscopy; PGs; phosphatidylglycerols; SDS; sodium dodecylsulfate; TOCSY; total correlation spectroscopyAntimicrobial peptides; Chemical shifts; Dynamics; HSQC wave; Membrane-binding residues; Membrane proteins; Micelles; NMR waves

NMR structure of the transmembrane and cytoplasmic domains of human CD4 in micelles by Marc Wittlich; Pallavi Thiagarajan; Bernd W. Koenig; Rudolf Hartmann; Dieter Willbold (pp. 122-127).

The human cluster determinant 4 (CD4) is a type I transmembrane glycoprotein involved in T-cell signalling. It is expressed primarily on the surface of T helper cells but also on subsets of memory and regulatory T lymphocytes (CD4⁺ cells). It serves as a coreceptor in T-cell receptor recognition of MHC II antigen complexes. Besides its cellular functions, CD4 serves as the main receptor for human immunodeficiency virus type I (HIV-1). During T-cell infection, the CD4 extracellular domain is bound by HIV-1 gp120, the viral surface glycoprotein, which triggers a number of conformational changes ultimately resulting in virion entry of the cell. Subsequently, CD4 is downregulated in infected cells by multiple strategies that involve direct interactions of the HIV-1 proteins VpU and Nef with the cytoplasmic part of CD4. In the present work, we describe the NOE-based solution structure of the transmembrane and cytoplasmic domains of the cystein-free variant of CD4 (CD4mut) in dodecylphosphocholine (DPC) micelles. Furthermore, we have characterized micelle-inserted CD4mut by paramagentic relaxation enhancement (PRE) agents and¹H-¹⁵N heteronuclear NOE data. CD4mut features a stable and well-defined transmembrane helix from M372 to V395 buried in the micellar core and a cytoplasmic helix ranging from A404 to L413. Experimental data suggest the amphipathic cytoplasmic helix to be in close contact with the micellar surface. The role of the amphipathic helix and its interaction with the micellar surface is discussed with respect to the biological function of the full-length CD4 protein.

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; 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

Micelle-bound structures and dynamics of the hinge deleted analog of melittin and its diastereomer: Implications in cell selective lysis byd-amino acid containing antimicrobial peptides by Rathi Saravanan; Anirban Bhunia; Surajit Bhattacharjya (pp. 128-139).

Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel-^dH containingd-amino acids [^dV5,^dV8,^dL11 and^dK16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H–D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel-^dH both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-^dH interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-^dH showed multiple β-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D₂O demonstrated a large difference in dynamics between Mel-H and Mel-^dH, whereby almost all backbone protons of Mel-^dH showed a much faster rate of exchange as compared to Mel-H. Proton T₁ relaxation had suggested a mobile backbone of Mel-^dH peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-^dH is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of thed-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface.

Keywords: Melittin; Antimicrobial peptide; AMPs; Helical AMPs; Antibiotic resistance

The impact of window functions on NMR-based paramagnetic relaxation enhancement measurements in membrane proteins by Wade D. Van Horn; Andrew J. Beel; Congbao Kang; Charles R. Sanders (pp. 140-149).

Though challenging, solution NMR spectroscopy allows fundamental interrogation of the structure and dynamics of membrane proteins. One major technical hurdle in studies of helical membrane proteins by NMR is the difficulty of obtaining sufficient long range NOEs to determine tertiary structure. For this reason, long range distance information is sometimes sought through measurement of paramagnetic relaxation enhancements (PRE) of NMR nuclei as a function of distance from an introduced paramagnetic probe. Current PRE interpretation is based on the assumption of Lorentzian resonance lineshapes. However, in order to optimize spectral resolution, modern multidimensional NMR spectra are almost always subjected to resolution-enhancement, leading to distortions in the Lorentizian peak shape. Here it is shown that when PREs are derived using peak intensities (i.e., peak height) and linewidths from both real and simulated spectra that were produced using a wide range of apodization/window functions, that there is little variation in the distances determined (<1 Å at the extremes). This indicates that the high degree of resolution enhancement required to obtain well-resolved spectra from helical membrane proteins is compatible with the use of PRE data as a source of distance restraints. While these conclusions are particularly important for helical membrane proteins, they are generally applicable to all PRE measurements made using resolution-enhanced data.

Keywords: Membrane protein; Paramagnetic relaxation enhancement; PRE; NMR; Apodization; Diacylglycerol kinase; KCNE1; Amyloid; Spin-labeling; Structure

A method for solution NMR structural studies of large integral membrane proteins: Reverse micelle encapsulation by Joseph M. Kielec; Kathleen G. Valentine; A. Joshua Wand (pp. 150-160).

The structural study of membrane proteins perhaps represents one of the greatest challenges of the post-genomic era. While membrane proteins comprise over 50% of current and potential drug targets, their structural characterization lags far behind that of soluble proteins. Nuclear magnetic resonance (NMR) offers great potential not only with respect to structural characterization of integral membrane proteins but may also provide the ability to study the details of small ligand interactions. However, the size limitations of solution NMR have restricted comprehensive structural characterization of membrane protein NMR structures to the relatively small β-barrel proteins or helical proteins of relatively simple topology. In an effort to escape the barriers presented by slow molecular reorientation of large integral membrane proteins solubilized by detergent micelles in water, we have adapted the reverse micelle encapsulation strategy originally developed for the study of large soluble proteins by solution NMR methods. Here we review a novel approach to the solubilization of large integral membrane proteins in reverse micelle surfactants dissolved in low viscosity alkane solvents. The procedure is illustrated with a 54kDa construct of the homotetrameric KcsA potassium channel.

Keywords: Abbreviations; AOT; sodium bis(2-ethylhexyl)sulfosuccinate; C; ₁₂; E; ₄; n; -dodecyl tetra ethylene glycol; CTAB; cetyltrimethylammonium bromide; DHAB; dihexadecyldimethylammonium bromide; DM; n-decyl-β-; d; -maltopyranoside; DPC; dodecylphosphocholine (foscholine); DTAB; dodecyltrimethylammonium bromide; HSQC; heteronuclear single quantum coherence; LDAO; lauryldimethylamine oxide; MCD; main chain directed; NAB; H; _N; –H; _α; –H; _β; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; T; ₂; spin–spin or transverse relaxation time constant; TOCSY; total correlation spectroscopy; TROSY; transverse relaxation optimized spectroscopyReverse micelles; Membrane proteins; Detergents; NMR; Potassium channel; Surfactants

Anesthetic effects on the structure and dynamics of the second transmembrane domains of nAChR α4β2 by Tanxing Cui; Christian G. Canlas; Yan Xu; Pei Tang (pp. 161-166).

Channel functions of the neuronal α4β2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na⁺ flux experiments confirmed that the second transmembrane domains (TM2) of α4 and β2 in 2:3 stoichiometry, (α4)₂(β2)₃, could form pentameric channels, whereas the α4 TM2 alone could not. The structure, topology, and dynamics of the α4 TM2 and (α4)₂(β2)₃ TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics.²H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively¹⁵N-labeled α4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12°±1° and 16°±1° relative to the bicelle normal for the α4 and (α4)₂(β2)₃ samples, respectively. Anesthetics changed the tilt angle of the α4 TM2 from 12°±1° to 14°±1°, but had only a subtle effect on the tilt angle of the (α4)₂(β2)₃ TM2. A small degree of wobbling motion of the helix axis occurred in the (α4)₂(β2)₃ TM2. In addition, a subset of the (α4)₂(β2)₃ TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10–⁴ s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions.

Keywords: Mechanisms of general anesthesia; Halothane; Isoflurane; Volatile anesthetics; PISEMA; Solid state NMR; nAChR; a4b2; Neuronal nAChR

Suppressed or recovered intensities analysis in site-directed¹³C NMR: Assessment of low-frequency fluctuations in bacteriorhodopsin and D85N mutants revisited by Hazime Saitô; Atsushi Kira; Tadashi Arakawa; Michikazu Tanio; Satoru Tuzi; Akira Naito (pp. 167-176).

The first proton transfer of bacteriorhodopsin (bR) occurs from the protonated Schiff base to the anionic Asp 85 at the central part of the protein in the L to M states. Low-frequency dynamics accompanied by this process can be revealed by suppressed or recovered intensities (SRI) analysis of site-directed¹³C solid-state NMR spectra of 2D crystalline preparations. First of all, we examined a relationship of fluctuation frequencies available from [1-¹³C]Val- and [3-¹³C]Ala-labeled preparations, by taking the effective correlation time of internal methyl rotations into account. We analyzed the SRI data of [1-¹³C]Val-labeled wild-type bR and D85N mutants, as a function of temperature and pH, respectively, based on so-far assigned peaks including newly assigned or revised ones. Global conformational change of the protein backbone, caused by neutralization of the anionic D85 by D85N, can be visualized by characteristic displacement of peaks due to the conformation-dependent¹³C chemical shifts. Concomitant dynamics changes if any, with fluctuation frequencies in the order of 10⁴ Hz, were evaluated by the decreased peak intensities in the B–C and D–E loops of D85N mutant. The resulting fluctuation frequencies, owing to subsequent, accelerated dynamics changes in the M-like state by deprotonation of the Schiff base at alkaline pH, were successfully evaluated based on the SRI plots as a function of pH, which were varied depending upon the extent of interference of induced fluctuation frequency with frequency of magic angle spinning or escape from such interference. Distinguishing fluctuation frequencies between the higher and lower than 10⁴ Hz is now possible, instead of a simple description of the data around 10⁴ Hz available from one-point data analysis previously reported.

Keywords: Low-frequency dynamics; SRI analysis; Site-directed 13C NMR; Bacteriorhodopsin; D85N mutant site-directed 13C NMR

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy by Michael F. Brown; Gilmar F.J. Salgado; Andrey V. Struts (pp. 177-193).

Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state²H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific²H labels have been introduced into the methyl groups of retinal and solid-state²H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent²H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all- trans retinal with the β4 strand of the E2 loop and the side chains of Glu¹²² and Trp²⁶⁵ within the binding pocket. The solid-state²H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.

Keywords: Abbreviations; bR; bacteriorhodopsin; CD; circular dichroism; DARR; dipolar-assisted rotational-resonance; DOPE; 1,2-dioleoyl-; sn; -glycero-3-phosphoethanolamine; EFG; electric field gradient; ESR; electron spin resonance; FTIR; Fourier transform infrared; GPCR; G protein-coupled receptor; HOOP; hydrogen-out-of-plane; 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; RMSD; root mean square deviation; RDC; residual dipolar coupling; RQC; residual quadrupolar couplingMolecular dynamics; G protein-coupled receptor; Membrane; Solid-state NMR; Retinal; Rhodopsin; Signal transduction; Vision

Nuclear magnetic resonance evidence for retention of a lamellar membrane phase with curvature in the presence of large quantities of the HIV fusion peptide by Charles M. Gabrys; Rong Yang; Christopher M. Wasniewski; Jun Yang; Christian G. Canlas; Wei Qiang; Yan Sun; David P. Weliky ⁎ (pp. 194-201).

The HIV fusion peptide (HFP) is a biologically relevant model system to understand virus/host cell fusion.²H and³¹P NMR spectroscopies were applied to probe the structure and motion of membranes with bound HFP and with a lipid headgroup and cholesterol composition comparable to that of membranes of host cells of HIV. The lamellar phase was retained for a variety of highly fusogenic HFP constructs as well as a non-fusogenic HFP construct and for the influenza virus fusion peptide. The lamellar phase is therefore a reasonable structure for modeling the location of HFP in lipid/cholesterol dispersions. Relative to no HFP, membrane dispersions with HFP had faster³¹P transverse relaxation and faster transverse relaxation of acyl chain²H nuclei closest to the lipid headgroups. Relative to no HFP, mechanically aligned membrane samples with HFP had broader³¹P signals with a larger fraction of unoriented membrane. The relaxation and aligned sample data are consistent with bilayer curvature induced by the HFP which may be related to its fusion catalytic function. In some contrast to the subtle effects of HFP on a host-cell-like membrane composition, an isotropic phase was observed in dispersions rich in phosphatidylethanolamine lipids and with bound HFP.

Keywords: HIV; Fusion; Peptide; NMR; Curvature; Membrane

A solid-state NMR study of changes in lipid phase induced by membrane-fusogenic LV-peptides by Prashant Agrawal; Suzanne Kiihne; Johan Hollander; Mathias Hofmann; Dieter Langosch; Huub de Groot (pp. 202-209).

Membrane fusion requires restructuring of lipid bilayers mediated by fusogenic membrane proteins. Peptides that correspond to natural transmembrane sequences or that have been designed to mimic them, such as low-complexity “Leu-Val” (LV) peptide sequences, can drive membrane fusion, presumably by disturbing the lipid bilayer structure. Here, we assess how peptides of different fusogenicity affect membrane structure using solid state NMR techniques. We find that the more fusogenic variants induce an unaligned lipid phase component and a large degree of phase separation as observed in³¹P 2D spectra. The data support the idea that fusogenic peptides accumulate PE in a non-bilayer phase which may be critical for the induction of fusion.

Keywords: Abbreviations; DOPS; 1,2-dioleoyl-sn-glycero-3-[phospho-; l; -serine]; DOPE; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; HBTU; [2-(1 H-benzotriazol-1-yl)-1,1,3,3- etramethyluronium hexafuorophosphate]; NBD-PE; N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexadecylphosphatidylethanolamine; NMP; N-methylpyrrolidinone; POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Rh-PE; N-(lissamine rhodamin B sulfonyl)hexadecylphosphatidyl-ethanolamine; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PS; phospatidylserine; SNARE; soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; TMS; transmembrane segments; TIS; triisopropyl silaneMembrane fusion; Transmembrane fusogenic polypeptide; ³¹; P solid state NMR

Solid-state²H and¹⁵N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation by Shidong Chu; Aaron T. Coey; Gary A. Lorigan (pp. 210-215).

Phospholamban (PLB) is an integral membrane protein regulating Ca²⁺ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca²⁺ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with²H and¹⁵N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs).²H and¹⁵N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD₃-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca²⁺-ATPase; thus, showing increased inhibition of Ca²⁺-ATPase.

Keywords: Abbreviations; CD; circular dichroism; CP-MAS; cross-polarization magic-angle-spinning; PLB; phospholamban; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; MLVs; multilamellar vesicles; SR; sarcoplasmic reticulum; SERCA; sarco(endo)plasmic reticulum calcium ATPasePhospholamban; Phospholipid membrane; Solid-state NMR; Dynamics

Partitioning, dynamics, and orientation of lung surfactant peptide KL₄ in phospholipid bilayers by Joanna R. Long; Frank D. Mills; Omjoy K. Ganesh; Vijay C. Antharam; R. Suzanne Farver (pp. 216-222).

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL₄ is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL₄ is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL₄ in phospholipid bilayers, we introduced CD₃-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via²H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL₄ in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL₄ lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL₄ relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL₄. The unusual secondary structure of KL₄ and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.

Keywords: Abbreviations; SP-B; surfactant protein B; RDS; respiratory distress syndrome; TM; transmembrane; NMR; nuclear magnetic resonance; CD; circular dichroism; FTIR; Fourier transform infrared spectroscopy; MLV; multilamellar vesicle; LUV; large unilamellar vesicle; L; _α; fluid lamellar phase; L; _β; gel lamellar phase; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylethanolamine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylglycerol; DPPC; 1,2-dipalmitoyl-; sn; -Glycero-3-Phosphocholine; P/L; peptide/lipid molar ratioKL; ₄; Sinapultide; Lucinactant; Lung surfactant; Surfactant protein B; Respiratory distress syndrome; Lipid bilayer; ²; H NMR; Leucine side chain dynamics

Cholesterol reduces pardaxin's dynamics—a barrel-stave mechanism of membrane disruption investigated by solid-state NMR by Ayyalusamy Ramamoorthy; Dong-Kuk Lee; Tennaru Narasimhaswamy; Ravi P.R. Nanga (pp. 223-227).

While high-resolution 3D structures reveal the locations of all atoms in a molecule, it is the dynamics that correlates the structure with the function of a biological molecule. The complete characterization of dynamics of a membrane protein is in general complex. In this study, we report the influence of dynamics on the channel-forming function of pardaxin using chemical shifts and dipolar couplings measured from 2D broadband-PISEMA experiments on mechanically aligned phospholipids bilayers. Pardaxin is a 33-residue antimicrobial peptide originally isolated from the Red Sea Moses sole, Pardachirus marmoratus, which functions via either a carpet-type or barrel-stave mechanism depending on the membrane composition. Our results reveal that the presence of cholesterol significantly reduces the backbone motion and the tilt angle of the C-terminal amphipathic helix of pardaxin. In addition, a correlation between the dynamics-induced heterogeneity in the tilt of the C-terminal helix and the membrane disrupting activity of pardaxin by the barrel-stave mechanism is established. This correlation is in excellent agreement with the absence of hemolytic activity for the derivatives of pardaxin. These results explain the role of cholesterol in the selectivity of the broad-spectrum of antimicrobial activities of pardaxin.

Keywords: Dynamic; Pardaxin; Membrane orientation; Barrel-stave; Antimicrobial peptide

Can antimicrobial peptides scavenge around a cell in less than a second? by Eduard Y. Chekmenev; Breanna S. Vollmar; Myriam Cotten (pp. 228-234).

Antimicrobial peptides, which play multiple host-defense roles, have garnered increased experimental focus because of their potential applications in the pharmaceutical and food production industries. While their mechanisms of action are richly debated, models that have been advanced share modes of peptide–lipid interactions that require peptide dynamics. Before the highly cooperative and specific events suggested in these models take place, peptides must undergo an important process of migration along the membrane surface and delivery from their site of binding on the membrane to the actual site of functional performance. This phenomenon, which contributes significantly to antimicrobial function, is poorly understood, largely due to a lack of experimental and computational tools needed to assess it. Here, we use¹⁵N solid-state nuclear magnetic resonance to obtain molecular level data on the motions of piscidin's amphipathic helices on the surface of phospholipid bilayers. The studies presented here may help contribute to a better understanding of the speed at which the events that lead to antimicrobial response take place. Specifically, from the perspective of the kinetics of cellular processes, we discuss the possibility that piscidins and perhaps many other amphipathic antimicrobial peptides active on the membrane surface may represent a class of fast scavengers rather than static polypeptides attached to the water–lipid interface.

Keywords: Abbreviations; AMPs; antimicrobial peptides; APD; antimicrobial peptide database; CSA; chemical shift anisotropy; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phosphoglycerate; E-field; electric-field; HPLC; high performance liquid chromatography; MIC; minimum inhibitory concentration; NMR; nuclear magnetic resonance; p1(or 3)-COO; ⁻; non-amidated piscidin 1 (or 3); p1(or 3)-NH; ₂; amidated piscidin 1 (or 3); PC; phosphatidylcholine; PDB; protein data bank; PG; phosphatidylglycerol; TFE; trifluoroethanolPiscidin; Structure–function relationship; Solid-state NMR; Peptide dynamics; Chemical shift anisotropy; Water-bilayer interface

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: A solid-state NMR study by Marise Ouellet; Normand Voyer; Michèle Auger (pp. 235-243).

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation,³¹P and²H NMR experiments were performed on 21-mer peptide-containing bicelles.³¹P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand,²H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding.¹⁵N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface.¹⁵N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally,¹³C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the¹³CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.

Keywords: Synthetic ion channel; Solid-state NMR spectroscopy; Membrane topology; Bicelle; Peptide dynamic; Lipid/peptide interaction

Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II by Alison Drechsler; Gregor Anderluh; Raymond S. Norton; Frances Separovic (pp. 244-251).

Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or “rafts” in PC membranes,³¹P and²H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T₂ relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.

Keywords: Abbreviations; CDCl; ₃; deuterated-chloroform; Chol; Cholesterol; CSA; chemical shift anisotropy; DMPC; dimyristoylphosphatidylcholine; EqtII; equinatoxin II; MAS; magic angle spinning NMR; MLV; multilamellar vesicles; POPC; palmitoyloleoylphosphatidylcholine; SDS; sodium dodecyl sulfate; SDS-PAGE; SDS-polyacrylamide gel electophoresis; SM; sphingomyelinEquinatoxin II; Lipid domain; Membrane toxin; Phospholipid bilayer; Protein-lipid interaction; Solid-state NMR

Solid state NMR analysis of peptides in membranes: Influence of dynamics and labeling scheme by Santi Esteban-Martín; Erik Strandberg; Jesús Salgado; Anne S. Ulrich (pp. 252-257).

The functional state of a membrane-active peptide is often defined by its conformation, molecular orientation, and its oligomeric state in the lipid bilayer. These “static” structural properties can be routinely studied by solid state NMR using isotope-labeled peptides. In the highly dynamic environment of a liquid crystalline biomembrane, however, the whole-body fluctuations of a peptide are also of paramount importance, although difficult to address and most often ignored. Yet it turns out that disregarding such motional averaging in calculating the molecular alignment from orientational NMR-constraints may give a misleading, if not false picture of the system. Here, we demonstrate that the reliability of a simplified static or an advanced dynamic data analysis depends critically on the choice of isotope labeling scheme used. Two distinctly different scenarios have to be considered. When the labels are placed on the side chains of a helical peptide (such as a CD₃- or CF₃-group attached to the C^αC^β bond), their nuclear spin interaction tensors are very sensitive to motional averaging. If this effect is not properly accounted for, the helix tilt angle tends to be severely underestimated. At the same time, the analysis of labels in the side chains allows to extract valuable dynamical information about whole-body fluctuations of the peptide helix in the membrane. On the other hand, the alternative labeling scheme where¹⁵N-labels are accommodated within the peptide backbone, will yield nearly correct helix tilt angles, irrespective as to whether dynamics are taken into account or not.

Keywords: Membrane-bound peptide; α-helices; Peptide orientation; Peptide dynamic; Whole body fluctuation; Isotope labeling scheme; Solid-state; ²; H-,; ¹⁹; F-,; ¹⁵; N-NMR; PISEMA; GALA; NMR tensor orientation

Solid-state NMR approaches to measure topological equilibria and dynamics of membrane polypeptides by Evgeniy Salnikov; Christopher Aisenbrey; Verica Vidovic; Burkhard Bechinger ⁎ (pp. 258-265).

Biological membranes are characterized by a high degree of dynamics. In order to understand the function of membrane proteins and even more of membrane-associated peptides, these motional aspects have to be taken into consideration. Solid-state NMR spectroscopy is a method of choice when characterizing topological equilibria, molecular motions, lateral and rotational diffusion as well as dynamic oligomerization equilibria within fluid phase lipid bilayers. Here we show and review examples where the¹⁵N chemical shift anisotropy, dipolar interactions and the deuterium quadrupolar splittings have been used to analyze motions of peptides such as peptaibols, antimicrobial sequences, Vpu, phospholamban or other channel domains. In particular, simulations of¹⁵N and²H-solid-state NMR spectra are shown of helical domains in uniaxially oriented membranes when rotation around the membrane normal or the helix long axis occurs.

Keywords: Abbreviations; bR; bacteriorhodopsin; CD; circular dichroism; CP; cross polarization; CSA; chemical shift anisotropy; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; MAS; magic angle spinning; NMR; nuclear magnetic resonance; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerolAlamethicin; Peptaibols; Antimicrobial peptide; Transmembrane; Alpha-helix; Topology; Hydrophobic mismatch; Tilt and rotational pitch angle; Azimuthal angle; Rotational diffusion; Lateral diffusion; Helix axis; Wobbling motion; Libration

A solid-state NMR study of the structure and dynamics of the myristoylated N-terminus of the guanylate cyclase-activating protein-2 by Stephan Theisgen; Holger A. Scheidt; Alvicler Magalhães; Tito J. Bonagamba; Daniel Huster (pp. 266-274).

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca²⁺ sensor protein. It plays a central role in shaping the photoreceptor light response and in light adaptation through the Ca²⁺-dependent regulation of the transmembrane retinal guanylate cyclase (GC). GCAP-2 is N-terminally myristoylated and the full activation of the GC requires this lipid modification. The structural and functional role of the N-terminus and particularly of the myristoyl moiety is currently not well understood. In particular, detailed structural information on the myristoylated N-terminus in the presence of membranes was not available. Therefore, we studied the structure and dynamics of a 19 amino acid peptide representing the myristoylated N-terminus of GCAP-2 bound to lipid membranes by solid-state NMR.¹³C isotropic chemical shifts revealed a random coiled secondary structure of the peptide. Peptide segments up to Ala₉ interact with the membrane surface. Order parameters for Cα and side chain carbons obtained from DIPSHIFT experiments are relatively low, suggesting high mobility of the membrane-associated peptide. Static²H solid-state NMR measurements show that the myristoyl moiety is fully incorporated into the lipid membrane. The parameters of the myristoyl moiety and the DMPC host membrane are quite similar. Furthermore, dynamic parameters (obtained from²H NMR relaxation rates) of the peptide's myristic acid chain are also comparable to those of the lipid chains of the host matrix. Therefore, the myristoyl moiety of the N-terminal peptide of GCAP-2 fills a similar conformational space as the surrounding phospholipid chains.

Keywords: Abbreviations; GC; guanylate cyclase; GCAP-2; guanylate cyclase-activating protein-2; NCS; neuronal calcium sensor; CP MAS; cross-polarization magic angle spinning; HetCor; heteronuclear correlation; DIPSHIFT; dipolar coupling and chemical shift; DMPC; 1,2-dimyristoylphosphocholineLipid modification; GCAP-2; Membrane-peptide interaction; MAS NMR; Order parameter

Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation by Alexander Vogel; Guido Reuther; Matthew B. Roark; Kui-Thong Tan; Herbert Waldmann; Scott E. Feller; Daniel Huster (pp. 275-285).

The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data.

Keywords: Abbreviations; DIPSHIFT; dipolar coupling and chemical shift; DMPC; 1,2-dimyristoyl-phosphocholine; FSLG; frequency-switched Lee-Goldburg; GTP; guanosine triphosphate; MAS; magic-angle spinning; MD; molecular dynamics; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser enhancement; PAS; principle axis system; TALOS; torsion angle likelihood obtained from shift and sequence similarity; TMS; tetramethylsilaneMembrane protein; Lipid modification; Structural dynamics; Replica exchange; Order parameter; Relaxation

Protein dynamics detected in a membrane-embedded potassium channel using two-dimensional solid-state NMR spectroscopy by Christian Ader; Olaf Pongs; Stefan Becker; Marc Baldus ⁎ (pp. 286-290).

We report longitudinal¹⁵N relaxation rates derived from two-dimensional (¹⁵N,¹³C) chemical shift correlation experiments obtained under magic angle spinning for the potassium channel KcsA-Kv1.3 reconstituted in multilamellar vesicles. Thus, we demonstrate that solid-state NMR can be used to probe residue-specific backbone dynamics in a membrane-embedded protein. Enhanced backbone mobility was detected for two glycine residues within the selectivity filter that are highly conserved in potassium channels and that are of core relevance to the filter structure and ion selectivity.

Keywords: Dynamics; Ion channel; MAS; Membrane; Protein; Solid-state NMR

Evidence of phosphatidylethanolamine and phosphatidylglycerol presence at the annular region of lactose permease of Escherichia coli by Laura Picas; M. Teresa Montero; Antoni Morros; J.L. Vázquez-Ibar; Jordi Hernández-Borrell (pp. 291-296).

Biochemical and structural work has revealed the importance of phospholipids in biogenesis, folding and functional modulation of membrane proteins. Therefore, the nature of protein–phospholipid interaction is critical to understand such processes. Here, we have studied the interaction of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl- sn-glycero-3-[phospho- rac-(1-glycerol)] (POPG) mixtures with the lactose permease (LacY), the sugar/H⁺ symporter from Escherichia coli and a well characterized membrane transport protein. FRET measurements between single-W151/C154G LacY reconstituted in a lipid mixture composed of POPE and POPG at different molar ratios and pyrene-labeled PE or PG revealed a different phospholipid distribution between the annular region of LacY and the bulk lipid phase. Results also showed that both PE and PG can be part of the annular region, being PE the predominant when the PE:PG molar ratio mimics the membrane of E. coli. Furthermore, changes in the thermotropic behavior of phospholipids located in this annular region confirm that the interaction between LacY and PE is stronger than that of LacY and PG. Since PE is a proton donor, the results obtained here are discussed in the context of the transport mechanism of LacY.

Keywords: Annular lipid; Lactose permease; Lipid–protein interaction; Förster resonance energy transfer

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1) by Karuppanan Muthusamy Kathir; Li Gao; Dakshinamurthy Rajalingam; Anna E. Daily; Sherri Brixey; Huimin Liu; Dan Davis; Paul Adams; Igor Prudovsky; Thallapuranam Krishnaswamy Suresh Kumar (pp. 297-302).

Human fibroblast growth factor (hFGF-1) is a ∼17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D¹H–¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.

Keywords: Abbreviations; hFGF-1; human fibroblast growth factor-1; Syt1; Synaptotagmin 1; HSQC; Heteronuclear Single Quantum Coherence; NMR; nuclear magnetic resonanceFibroblast growth factor; Secretion; Non-classical; Synaptotagmin; Lipid binding

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BBA - Biomembranes (v.1798, #2)