Membrane-bound Conformation Research Articles

Membrane-Bound Conformation of Peptaibols with Methyl-Deuterated α-Amino Isobutyric Acids by 2H Magic Angle Spinning Solid-State NMR Spectroscopy

We present the use of 2H magic-angle spinning (MAS) NMR on methyl-deuterated alpha-amino isobutyric acid (Aib) as a new method to obtain fast and accurate structural constraints on peptaibols in membrane-bound environments. Using nonoriented vesicle-reconstituted samples we avoid the delicate preparation of oriented samples, and the use of MAS ensures high sensitivity and thereby very fast acquisition of experimental spectra. Furthermore, given the high content ( approximately 40%) of Aib in peptaibols and the fact that the amino acid Aib may be synthesized from cheap starting materials, even in the case of 2H, 13C, or 15N labeling, this method is ideally suited for studies of the membrane-bound conformation of peptaibols.

Defining the structure of membrane-bound human blood coagulation factor Va.

Blood coagulation factor (F) Va is the essential protein cofactor to the serine protease FXa. Factor Va stimulates the thrombin-to-prothrombin conversion by the prothrombinase complex, by at least five orders of magnitude. Factor Va binds with very high affinity to phosphatidylserine containing phospholipid membranes, which allows the visualization of its membrane-bound state by transmission electron microscopy (EM). In this paper we present an averaged three-dimensional structure of FVa molecules attached to phosphatidylserine containing lipid tubes, as determined by EM and single particle analysis. The low-resolution FVa three-dimensional structure is compared with the available atomic models for FVa. The experimental data are combined with the most suitable atomic model and a membrane-bound FVaEM model is proposed that best fits the protein density defined by EM. In the FVaEM model, the C1 and C2 membrane-binding domains are juxtaposed onto the membrane surface and the model geometries indicate a deeper insertion of both C domains into the lipid bilayer than has been previously suggested. The present structure is a first step towards a higher-resolution experimental structure of a human FVa molecule in its membrane-bound conformation, allowing the visualization of individual domains within FVa and its association with the membrane.

The role of hydrophobic interactions in positioning of peripheral proteins in membranes

BackgroundThree-dimensional (3D) structures of numerous peripheral membrane proteins have been determined. Biological activity, stability, and conformations of these proteins depend on their spatial positions with respect to the lipid bilayer. However, these positions are usually undetermined.ResultsWe report the first large-scale computational study of monotopic/peripheral proteins with known 3D structures. The optimal translational and rotational positions of 476 proteins are determined by minimizing energy of protein transfer from water to the lipid bilayer, which is approximated by a hydrocarbon slab with a decadiene-like polarity and interfacial regions characterized by water-permeation profiles. Predicted membrane-binding sites, protein tilt angles and membrane penetration depths are consistent with spin-labeling, chemical modification, fluorescence, NMR, mutagenesis, and other experimental studies of 53 peripheral proteins and peptides. Experimental membrane binding affinities of peripheral proteins were reproduced in cases that did not involve a helix-coil transition, specific binding of lipids, or a predominantly electrostatic association. Coordinates of all examined peripheral proteins and peptides with the calculated hydrophobic membrane boundaries, subcellular localization, topology, structural classification, and experimental references are available through the Orientations of Proteins in Membranes (OPM) database.ConclusionPositions of diverse peripheral proteins and peptides in the lipid bilayer can be accurately predicted using their 3D structures that represent a proper membrane-bound conformation and oligomeric state, and have membrane binding elements present. The success of the implicit solvation model suggests that hydrophobic interactions are usually sufficient to determine the spatial position of a protein in the membrane, even when electrostatic interactions or specific binding of lipids are substantial. Our results demonstrate that most peripheral proteins not only interact with the membrane surface, but penetrate through the interfacial region and reach the hydrocarbon interior, which is consistent with published experimental studies.

Diffraction-Based Density Restraints for Membrane and Membrane-Peptide Molecular Dynamics Simulations

We have recently shown that current molecular dynamics (MD) atomic force fields are not yet able to produce lipid bilayer structures that agree with experimentally-determined structures within experimental errors. Because of the many advantages offered by experimentally validated simulations, we have developed a novel restraint method for membrane MD simulations that uses experimental diffraction data. The restraints, introduced into the MD force field, act upon specified groups of atoms to restrain their mean positions and widths to values determined experimentally. The method was first tested using a simple liquid argon system, and then applied to a neat dioleoylphosphatidylcholine (DOPC) bilayer at 66% relative humidity and to the same bilayer containing the peptide melittin. Application of experiment-based restraints to the transbilayer double-bond and water distributions of neat DOPC bilayers led to distributions that agreed with the experimental values. Based upon the experimental structure, the restraints improved the simulated structure in some regions while introducing larger differences in others, as might be expected from imperfect force fields. For the DOPC-melittin system, the experimental transbilayer distribution of melittin was used as a restraint. The addition of the peptide caused perturbations of the simulated bilayer structure, but which were larger than observed experimentally. The melittin distribution of the simulation could be fit accurately to a Gaussian with parameters close to the observed ones, indicating that the restraints can be used to produce an ensemble of membrane-bound peptide conformations that are consistent with experiments. Such ensembles pave the way for understanding peptide-bilayer interactions at the atomic level.

Membrane-Bound Conformation and Topology of the Antimicrobial Peptide Tachyplesin I by Solid-State NMR

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.

Structure of Tightly Membrane-Bound Mastoparan-X, a G-Protein-Activating Peptide, Determined by Solid-State NMR

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C–C, N–C α C β , and N–C α –C′, and interresidue C α –C α C β , N–C α C β , and N–C′–C α correlation experiments. The backbone torsion angles were predicted from the chemical shifts of 13C′, 13C α , 13C β , and 15N signals with the aid of protein NMR database programs. In addition, two 13C– 13C and three 13C– 15N distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 Å. Peptide MP-X in the bilayer-bound state formed an amphiphilic α-helix for residues Trp 3−Leu 14 and adopted an extended conformation for Asn 2. This membrane-bound conformation is discussed in relation to the peptide’s activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.

Monitoring ion channel conformations in membranes utilizing a novel dual fluorescence quenching approach

The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics, and function of membrane-spanning channels. We have analyzed the localization of the functionally important tryptophan residues of the membrane-bound channel and non-channel conformations of gramicidin utilizing a novel dual fluorescence quenching approach [G.A. Caputo, E. London, Biochemistry 42 (2003) 3265–3274]. In this paper, we show for the first time that the dual quenching approach is applicable to multiple tryptophan containing functional ion channel peptides such as gramicidin. Importantly, dual quenching is found to be sensitive to the membrane-bound conformations of this important model ion channel.

Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membrane-bound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic alpha-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic alpha-helix (3.7 +/- 0.1 residues per turn and 97 +/- 3.0 degrees, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin.

Membrane-bound Conformation of M13 Major Coat Protein: A STRUCTURE VALIDATION THROUGH FRET-DERIVED CONSTRAINTS

M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or Förster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight alpha-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.

Lipid Membranes Modulate the Structure of Islet Amyloid Polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is thought to play an important role in the pathogenesis of type II diabetes. Despite a growing body of evidence implicating membrane interaction in IAPP toxicity, the membrane-bound form has not yet been well characterized. Here we used circular dichroism (CD) and fluorescence spectroscopy to investigate the molecular details of the interaction of IAPP with lipid membranes of varying composition. In the presence of membranes containing negatively charged phosphatidylserine (PS), we observed significant acceleration in the formation of IAPP aggregates. This acceleration is strongly modulated by the PS concentration and ionic strength, and is also observed at physiologically relevant PS concentrations. CD spectra of IAPP obtained immediately after the addition of membranes containing PS revealed features characteristic of an alpha-helical conformation approximately approximately 15-19 residues in length. After a longer incubation with membranes, IAPP gave rise to CD spectra characteristic of a beta-sheet conformation. Taken together, our CD and fluorescence data indicate that conditions that promote weakly stable alpha-helical conformations may promote IAPP aggregation. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.

Conformational States and Thermodynamics of α-Lactalbumin Bound to Membranes: A Case Study of the Effects of pH, Calcium, Lipid Membrane Curvature and Charge

The study of the conformational changes of bovine alpha-lactalbumin, switching from soluble states to membrane-bound states, deepens our knowledge of the behaviour of amphitropic proteins. The binding and the membrane-bound conformations of alpha-lactalbumin are highly sensitive to environmental factors, like calcium and proton concentrations, curvature and charge of the lipid membrane. The interactions between the protein and the membrane result from a combination of hydrophobic and electrostatic interactions and the respective weights of these interactions depend on the physicochemical conditions. As inferred by macroscopic as well as residue-level methods, the conformations of the membrane-bound protein range from native-like to molten globule-like states. However, the regions anchoring the protein to the membrane are similar and restricted to amphiphilic alpha-helices. H/(2)H-exchange experiments also yield residue-level data that constitute comprehensive information providing a new point of view on the thermodynamics of the interactions between the protein and the membrane.

Structural Basis of Citrate-dependent and Heparan Sulfate-mediated Cell Surface Retention of Cobra Cardiotoxin A3

Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.

Computational approaches to nanobiotechnology: probing the interaction of syntheticmolecules with phospholipid bilayers via a coarse grain model

This paper illustrates the use of computer simulation in understanding the interaction ofnano-scale synthetic molecules with cellular membranes, which are themselves a fewnanometres thick. Specifically, the interaction of a membrane-bound synthetic moleculewith a model membrane has been studied using so-called coarse-grain (CG) moleculardynamics (MD). The reported CG-MD simulations have been carried out for apore-promoting hydraphile molecule in a dimyristoylphosphatidylcholine (DMPC) lipidbilayer. Analysis of the CG-MD simulation provides insights into the nature of theinteraction of the hydraphile with the lipid molecules and how an initially fully extendedtrans-membrane hydraphile adjusts its end-to-end distance to match the bilayer thickness.The equilibrium membrane-bound hydraphile conformation observed in the CG-MDsimulation agrees well with deductions based on experimental observations. The success ofthe CG-MD approach in the present example suggests that the methodology couldbe used as a design tool in related nano-science and engineering applications.

Rab3D and annexin A2 play a role in regulated secretion of vWF, but not tPA, from endothelial cells.

von-Willebrand factor (vWF) and tissue-type plasminogen activator (tPA) are products of endothelial cells acutely released into the vasculature following cell activation. Both factors are secreted after intraendothelial Ca2+ mobilization, but exhibit opposing physiological effects with vWF inducing coagulation and tPA triggering fibrinolysis. To identify components that could regulate differentially the release of pro- and antithrombogenic factors, we analyzed the contribution of Rab3D and the annexin A2/S100A10 complex, proteins implicated in exocytotic events in other systems. We show that mutant Rab3D proteins interfere with the formation of bona fide Weibel-Palade bodies (WPbs), the principal storage granules of multimeric vWF, and consequently the acute, histamine-induced release of vWF. In contrast, neither appearance nor exocytosis of tPA storage granules is affected. siRNA-mediated downregulation of annexin A2/S100A10 and disruption of the complex by microinjection of peptide competitors result in a marked reduction in vWF but not tPA secretion, without affecting the appearance of WPbs. This indicates that distinct mechanisms underlie the acute secretion of vWF and tPA, enabling endothelial cells to fine-regulate the release of thrombogenic and fibrinolytic factors.

Effect of pH and ionic strength on the cytolytic toxin Cyt1A: a fluorescence spectroscopy study

Cyt1A is a cytolytic toxin produced by Bacillus thuringiensis var. israelensis. Due to its toxicity in vivo against mosquitoes and black flies, it is used as an environmentally friendly insecticide, although its mode of action is not completely understood. The toxin is membrane-active, but its membrane-bound conformation is unknown. In the absence of direct structural data, fluorescence spectroscopy was used to obtain indirect information on Cyt1A conformation changes in the environment mimicking the vicinity of the lipid membrane (lower pH and increased ionic strength). With decreasing pH, Cyt1A's surface hydrophobicity increased, which is consistent with an increased interaction with model membranes at low pH values, as observed previously. The p K a value of this conformation change is 4.4±0.1. Intrinsic tryptophan fluorescence decreased with decreasing pH, and the p K a value was the same as the one determined with synthetic probes. The protein has two types of hydrophobic binding sites, and at low pH these sites bind more probe molecules (bis-ANS) with a higher affinity than at pH 7.4. When bound to the lipid, the toxin exhibited conformation similar to the molten-globule state and showed some characteristics also observed at low pH. However, the conformation of the lipid-bound toxin did not depend on pH. Neutral salts like NaCl and KCl induced conformational changes at neutral pH, but not at low pH. These changes were most probably due to specific interactions of the salt ions with the charged amino acids on the protein surface rather than due to general effects such as Hofmeister and Debye–Hückel. Our results might contribute to elucidating the mode of action of Cyt1A, and perhaps also to improving the formulation of the insecticidal preparations.

The Interaction of Peripheral Proteins and Membranes Studied with α-Lactalbumin and Phospholipid Bilayers of Various Compositions

To characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine alpha-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMR-monitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent alpha-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of alpha-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane.

The Membrane-bound Conformation of α-Lactalbumin Studied by NMR-monitored 1H Exchange

The interaction of bovine α-lactalbumin (BLA) with negatively charged phospholipid bilayers was studied by NMR monitored 1H exchange to characterize the conformational transition that enables a water-soluble protein to associate with and partially insert into a membrane. BLA was allowed to exchange in deuterated buffer in the absence (reference) and the presence (membrane-bound) of acidic liposomes at pH 4.5, experimental conditions that allow efficient protein–membrane interaction. After adjusting the pH to 6.0, to dissociate the protein from the membrane, reference and membrane-released samples of BLA were analysed by (F1) band-selective homonuclear decoupled total correlation spectroscopy in the αH–NH region. The overall exchange behaviour of the membrane-bound state is molten globule-like, suggesting an overall destabilization of the polypeptide. Nevertheless, the backbone amide protons of residues R10, L12, C77, K94, K98, V99 and W104 show significant protection against solvent exchange in the membrane-bound protein. We propose a mechanism for the association of BLA with negatively charged membranes that includes initial protonation of acidic side-chains at the membrane interface, and formation of an interacting site with the membrane which involves helixes A and C. In the next step these helices would slide away from each other, adopting a parallel orientation to the membrane, and would rotate to maximize the interaction between their hydrophobic residues and the lipid bilayer.

Solid-State Nuclear Magnetic Resonance Evidence for an Extended β Strand Conformation of the Membrane-Bound HIV-1 Fusion Peptide

Solid-state nuclear magnetic resonance (NMR) spectroscopy was applied to the membrane-bound form of a synthetic peptide representing the 23-residue N-terminal fusion peptide domain of the HIV-1 gp41 envelope glycoprotein. 1D solid-state NMR line width measurements of singly 13C carbonyl labeled peptides showed that a significant population of the membrane-bound peptide is well-structured in its N-terminal and central regions while the C-terminus has more disorder. There was some dependence of line width on lipid composition, with narrower line widths and hence greater structural order observed for a lipid composition comparable to that found in the virus and its target T cells. In the more ordered N-terminal and central regions of the peptide, the 13C carbonyl chemical shifts are consistent with a nonhelical membrane-bound conformation. Additional evidence for a beta strand membrane-bound conformation was provided by analysis of 2D rotor-synchronized magic angle spinning NMR spectra of doubly 13C carbonyl labeled peptides. Lipid mixing and aqueous contents leakage assays were applied to demonstrate the fusogenicity of the peptide under conditions comparable to those used for the solid-state NMR sample preparation.

Vibrational circular dichroism of gramicidin D in vesicles and micelles.

The vibrational circular dichroism (VCD) and absorption spectra of gramicidin D in three model membranes (dioctadecyldimethylammonium chloride vesicles, dimyristoyl-phosphatidylcholine vesicles, and sodium dodecyl sulfate micelles) are presented. The absorption and VCD spectra suggest that the stable gramicidin D conformation in the model membranes is different from those in organic solvents. The presence of cations does not change the membrane-bound conformation of gramicidin D.

Vibrational circular dichroism of gramicidin D in vesicles and micelles

The vibrational circular dichroism (VCD) and absorption spectra of gramicidin D in three model membranes (dioctadecyldimethylammonium chloride vesicles, dimyristoyl-phosphatidylcholine vesicles, and sodium dodecyl sulfate micelles) are presented. The absorption and VCD spectra suggest that the stable gramicidin D conformation in the model membranes is different from those in organic solvents. The presence of cations does not change the membrane-bound conformation of gramicidin D. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 336–340, 2001