Molecular dynamics simulations predict a tilted orientation for the helical region of dynorphin A(1-17) in dimyristoylphosphatidylcholine bilayers

The structural properties of the endogenous opioid peptide dynorphin A(1-17) (DynA), a potential analgesic, were studied with molecular dynamics simulations in dimyristoylphosphatidylcholine bilayers. Starting with the known NMR structure of the peptide in dodecylphosphocholine micelles, the N-terminal helical segment of DynA (encompassing residues 1-10) was initially inserted in the bilayer in a perpendicular orientation with respect to the membrane plane. Parallel simulations were carried out from two starting structures, systems A and B, that differ by 4 A in the vertical positioning of the peptide helix. The complex consisted of approximately 26,400 atoms (dynorphin + 86 lipids + approximately 5300 waters). After >2 ns of simulation, which included >1 ns of equilibration, the orientation of the helical segment of DynA had undergone a transition from parallel to tilted with respect to the bilayer normal in both the A and B systems. When the helix axis achieved a approximately 50 degrees angle with the bilayer normal, it remained stable for the next 1 ns of simulation. The two simulations with different starting points converged to the same final structure, with the helix inserted in the bilayer throughout the simulations. Analysis shows that the tilted orientation adopted by the N-terminal helix is due to specific interactions of residues in the DynA sequence with phospholipid headgroups, water, and the hydrocarbon chains. Key elements are the "snorkel model"-type interactions of arginine side chains, the stabilization of the N-terminal hydrophobic sequence in the lipid environment, and the specific interactions of the first residue, Tyr. Water penetration within the bilayer is facilitated by the immersed DynA, but it is not uniform around the surface of the helix. Many water molecules surround the arginine side chains, while water penetration near the helical surface formed by hydrophobic residues is negligible. A mechanism of receptor interaction is proposed for DynA, involving the tilted orientation observed from these simulations of the peptide in the lipid bilayer.     

Two-dimensional solid-state NMR reveals two topologies of sarcolipin in oriented lipid bilayers

Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B(1-25)). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B(1-25) was compared with that of a truncated homodimer (dSP-B(8-25)) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at approximately 500 cm(-1) indicated a stable, although highly strained disulfide conformation with a chi(CS-SC) dihedral angle of +/-10 degrees for the dSP-B(1-25) dimer. In contrast, the truncated dimer dSP-B(8-25) exhibited a series of disulfide conformations with the chi(CS-SC) dihedral angle taking on values of either +/-30 degrees or 85+/-20 degrees . For conformations with chi(CS-SC) close to the +/-90 degrees value, the Raman spectra of the 8-25 truncated dimers exhibited chi(SS-CC) dihedral angles of 90/180 degrees and 20-30 degrees . In the presence of a lipid mixture, both constructs showed a nu(S-S) band at approximately 488 cm(-1), corresponding to a chi(CS-SC) dihedral angle of +/-10 degrees . Polarized infrared spectroscopy was also used to determine the orientation of the helix and beta-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of approximately 60-70 degrees to the surface normal, while the beta structure had approximately 40 degrees tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

Transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations.

To probe the fundamentals of membrane/protein interactions, all-atom multi-nanosecond molecular dynamics simulations were conducted on a single transmembrane poly(32)alanine helix in a fully solvated dimyristoyphosphatidylcholine (DMPC) bilayer. The central 12 residues, which interact only with the lipid hydrocarbon chains, maintained a very stable helical structure. Helical regions extended beyond these central 12 residues, but interactions with the lipid fatty-acyl ester linkages, the lipid headgroups, and water molecules made the helix less stable in this region. The C and N termini, exposed largely to water, existed as random coils. As a whole, the helix tilted substantially, from perpendicular to the bilayer plane (0 degree) to a 30 degrees tilt. The helix experienced a bend at its middle, and the two halves of the helix at times assumed substantially different tilts. Frequent hydrogen bonding, of up to 0.7 ns in duration, occurred between peptide and lipid molecules. This resulted in correlated translational diffusion between the helix and a few lipid molecules. Because of the large variation in lipid conformation, the lipid environment of the peptide was not well defined in terms of "annular" lipids and on average consisted of 18 lipid molecules. When compared with a "neat" bilayer without peptide, no significant difference was seen in the bilayer thickness, lipid conformations or diffusion, or headgroup orientation. However, the lipid hydrocarbon chain order parameters showed a significant decrease in order, especially in those methylene groups closest to the headgroup.     

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.     

Revisiting Hydrophobic Mismatch with Free Energy Simulation Studies of Transmembrane Helix Tilt and Rotation

Protein-lipid interaction and bilayer regulation of membrane protein functions are largely controlled by the hydrophobic match between the transmembrane (TM) domain of membrane proteins and the surrounding lipid bilayer. To systematically characterize responses of a TM helix and lipid adaptations to a hydrophobic mismatch, we have performed a total of 5.8-μs umbrella sampling simulations and calculated the potentials of mean force (PMFs) as a function of TM helix tilt angle under various mismatch conditions. Single-pass TM peptides called WALPn (n = 16, 19, 23, and 27) were used in two lipid bilayers with different hydrophobic thicknesses to consider hydrophobic mismatch caused by either the TM length or the bilayer thickness. In addition, different flanking residues, such as alanine, lysine, and arginine, instead of tryptophan in WALP23 were used to examine their influence. The PMFs, their decomposition, and trajectory analysis demonstrate that 1), tilting of a single-pass TM helix is the major response to a hydrophobic mismatch; 2), TM helix tilting up to ∼10° is inherent due to the intrinsic entropic contribution arising from helix precession around the membrane normal even under a negative mismatch; 3), the favorable helix-lipid interaction provides additional driving forces for TM helix tilting under a positive mismatch; 4), the minimum-PMF tilt angle is generally located where there is the hydrophobic match and little lipid perturbation; 5), TM helix rotation is dependent on the specific helix-lipid interaction; and 6), anchoring residues at the hydrophilic/hydrophobic interface can be an important determinant of TM helix orientation.     

Toward elucidating the membrane topology of helix two of the colicin E1 channel domain

The membrane-bound closed state of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane fluorophore attached to each single cysteine residue within helix 2 of each mutant protein. The fluorescence properties of the bimane fluorophore were measured for the membrane-associated form of the closed channel and included fluorescence emission maximum, fluorescence anisotropy, apparent polarity, surface accessibility, and membrane bilayer penetration depth. The fluorescence data show that helix 2 is an amphipathic alpha-helix that is situated parallel to the membrane surface, but it is less deeply embedded within the bilayer interfacial region than is helix 1 in the closed channel. A least squares fit of the various data sets to a harmonic wave function indicated that the periodicity and angular frequency for helix 2 in the membrane-bound state are typical for an amphipathic alpha-helix (3.8 +/- 0.1 residues per turn and 94 +/- 4 degrees, respectively) that is located at an interfacial region of a membrane bilayer. Dual quencher analysis also revealed that helix 2 is peripherally membrane associated, with one face of the helix dipping into the interfacial region of the lipid bilayer and the other face projecting outwardly into the aqueous solvent. Finally, our data show that helices 1 and 2 remain independent helices upon membrane association with a short connector link (Tyr(363)-Gly(364)) and that short amphipathic alpha-helices participate in the formation of a lipid-dependent, toroidal pore for this colicin.     

Organotin-induced hemolysis,shape transformation and intramembranous aggregates in human erythrocytes

Organotin compounds examined in this study exhibited a relative order of potency for induction of in vitro hemolysis in human erythrocytes as follows: tri-n-butyltin > tri-n propyltin > tetra-n-butyltin > triphenyltin chloride > tri-n-ethyltin bromide > dibutyltin dichloride > stannous chloride > tri-n-methyltin chloride = butyltin chloride dihydroxide. All of the organotin compounds induced erythrocyte shape transformation from the normal discocyte to an echinocyte and, in addition, triphenyltin chloride, tetra-n-butyltin and tri-n-ethyltin bromide also elicited stomatocyte formation at higher concentrations. Select organotin compounds also formed tin-containing aggregates within the plasma membrane. The relative order of effectiveness for organotin induction of intramembranous aggregates was tri-n-butyltin > tri-npropyltin > tetra-n-butyltin > tri-n-ethyltin bromide, which was based upon the lowest concentration at which they were observed. These results support the previously suggested theory that organotins are membrane effectors because of their comparatively high hydrophobic, lipid partitioning properties. The relatively lipophilic compound, triphenyltin chloride, appeared to be anomalous because it did not readily promote hemolysis or induce the formation of intramembranous aggregates in human erythrocytes. A log-linear statistical model demonstrated an association of hemolysis with both tri-n-butyltin aggregate formation and shape transformation. Select organotin compounds should be useful probes in membrane studies because of their numerous effects.Abbreviations DBT dibutylin dichloride - MBT butyltinchloride dihydroxide - SnCl₂ stannous chloride - TBT tri-n-butyltin - TET tri-n-ethyltin bromide - TMT tri-n-methyltin chloride - TPhT triphenyltin chloride - TPT tri-n-propyltin - TTBT tetra-n-butyltin     

Transmembrane four-helix bundle of influenza A M2 protein channel: structural implications from helix tilt and orientation.

The transmembrane portion of the M2 protein from the Influenza A virus has been studied in hydrated dimyristroylphosphotidylcholine lipid bilayers with solid-state NMR. Orientational constraints were obtained from isotopically labeled peptide samples mechanically aligned between thin glass plates. 15N chemical shifts from single site labeled samples constrain the molecular frame with respect to the magnetic field. When these constraints are applied to the peptide, modeled as a uniform alpha-helix, the tilt of the helix with respect to the bilayer normal was determined to be 33 degrees +/- 3 degrees. Furthermore, the orientation about the helix axis was also determined within an error of +/- 30 degrees. These results imply that the packing of this tetrameric protein is in a left-handed four-helix bundle. Only with such a large tilt angle are the hydrophilic residues aligned to the channel axis.     

The conformation of the pore region of the M2 proton channel depends on lipid bilayer environment

The M2 protein from influenza A virus is a 97-amino-acid protein with a single transmembrane helix that forms proton-selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix tilt, with proposed tilts ranging from approximately 15 degrees to 38 degrees . An important issue for understanding the structure of M2TM is the role of peptide-lipid interactions in the stabilization of the lipid bilayer bound tetramer. Here, we labeled the N terminus of M2TM with a nitroxide and studied the tetramer reconstituted into lipid bilayers of different thicknesses using EPR spectroscopy. Analyses of spectral changes provide evidence that the lipid bilayer does influence the conformation. The structural plasticity displayed by M2TM in response to membrane composition may be indicative of functional requirements for conformational change. The various structural models for M2TM proposed to date--each defined by a different set of criteria and in a different environment--might provide snapshots of the distinct conformational states sampled by the protein.     

Defining the transmembrane helix of M2 protein from influenza A by molecular dynamics simulations in a lipid bilayer

Integral membrane proteins containing at least one transmembrane (TM) alpha-helix are believed to account for between 20% and 30% of most genomes. There are several algorithms that accurately predict the number and position of TM helices within a membrane protein sequence. However, these methods tend to disagree over the beginning and end residues of TM helices, posing problems for subsequent modeling and simulation studies. Molecular dynamics (MD) simulations in an explicit lipid and water environment are used to help define the TM helix of the M2 protein from influenza A virus. Based on a comparison of the results of five different secondary structure prediction algorithms, three different helix lengths (an 18mer, a 26mer, and a 34mer) were simulated. Each simulation system contained 127 POPC molecules plus approximately 3500-4700 waters, giving a total of approximately 18,000-21,000 atoms. Two simulations, each of 2 ns duration, were run for the 18mer and 26mer, and five separate simulations were run for the 34mer, using different starting models generated by restrained in vacuo MD simulations. The total simulation time amounted to 11 ns. Analysis of the time-dependent secondary structure of the TM segments was used to define the regions that adopted a stable alpha-helical conformation throughout the simulation. This analysis indicates a core TM region of approximately 20 residues (from residue 22 to residue 43) that remained in an alpha-helical conformation. Analysis of atomic density profiles suggested that the 18mer helix revealed a local perturbation of the lipid bilayer. Polar side chains on either side of this region form relatively long-lived H-bonds to lipid headgroups and water molecules.     

Three-dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometry

The solution conformation of acyl carrier protein from Escherichia coli (77 residues) has been determined on the basis of 423 interproton-distance restraints and 32 hydrogen-bonding restraints derived from NMR measurements. A total of nine structures were computed using a hybrid approach combining metric matrix distance geometry and dynamic simulated annealing. The polypeptide fold is well defined with an average backbone atomic root-mean-square difference of 0.20 +/- 0.03 nm between the final nine converged structures and the mean structure obtained by averaging their coordinates. The principal structural motif is composed of three helices: 1 (residues 3-12), 2 (residues 37-47) and 4 (residues 65-75) which line a hydrophobic cavity. Helices 2 and 4 are approximately parallel to each other and anti-parallel at an angle of approximately equal to 150 degrees to helix 1. The smaller helix 3 (residues 56-63) is at an angle of approximately equal to 100 degrees to helix 4.     

Measurement of thermodynamic parameters for hydrophobic mismatch 1: self-association of a transmembrane helix

Membrane partitioning and self-association of transmembrane helices are crucial thermodynamic steps for membrane protein folding, although experimental difficulties have hampered quantitative estimations of related thermodynamic parameters, especially in lipid bilayer environments. This article reports for the first time, the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the formation of the antiparallel dimer of the inert hydrophobic model transmembrane helix X-(AALALAA)(3)-Y (X = 7-nitro-2-1, 3-benzoxadiazol-4-yl (NBD) and Y = NH(2) (I) or X = Ac and Y = NHCH(2)CH(2)-S-N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) (II)) in dimonounsaturated phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 5-55 degrees C, as evaluated by fluorescence resonance energy transfer from I to II. Stronger dimerization was observed in thicker membranes and at lower temperatures (DeltaG = -9 to -26 kJ mol(-)(1)), driven by large negative DeltaH values (-18 to -80 kJ mol(-)(1)). Fourier transform infrared-polarized spectroscopy revealed that the peptide formed a stable transmembrane helix with an orientation angle of approximately 15 degrees in all bilayers without significant effects on lipid structures, suggesting that the depth to which the helix termini penetrate changes depending on the degree of hydrophobic mismatch. The enthalpy changes for helix-helix interactions can be well explained by the electrostatic interactions between helix macrodipoles in different dielectric environments. The new concept of dipole-dipole interaction as a basic driving force of helix dimerization will become a basis for understanding the structural and functional modifications in response to hydrophobic mismatch.     

Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy

The structure of the membrane anchor domain (VpuMA) of the HIV-1-specific accessory protein Vpu has been investigated in solution and in lipid bilayers by homonuclear two-dimensional and solid-state nuclear magnetic resonance spectroscopy, respectively. Simulated annealing calculations, using the nuclear Overhauser enhancement data for the soluble synthetic peptide Vpu1-39 (positions Met-1-Asp-39) in an aqueous 2,2,2-trifluoroethanol (TFE) solution, afford a compact well-defined U-shaped structure comprised of an initial turn (residues 1-6) followed by a linker (7-9) and a short helix on the N-terminal side (10-16) and a further longer helix on the C-terminal side (22-36). The side chains of the two aromatic residues (Trp-22 and Tyr-29) in the longer helix are directed toward the center of the molecule around which the hydrophobic core of the folded VpuMA is positioned. As the observed solution structure is inconsistent with the formation of ion-conductive membrane pores defined previously for VpuMA in planar lipid bilayers, the isolated VpuMA domain as peptide Vpu1-27 was investigated in oriented phospholipid bilayers by proton-decoupled 15N cross polarization solid-state NMR spectroscopy. The line widths and chemical shift data of three selectively 15N-labeled peptides are consistent with a transmembrane alignment of a helical polypeptide. Chemical shift tensor calculations imply that the data sets are compatible with a model in which the nascent helices of the folded solution structure reassemble to form a more regular linear alpha-helix that lies parallel to the bilayer normal with a tilt angle of 相似文献   

Use of a new label, (13)==(18)O, in the determination of a structural model of phospholamban in a lipid bilayer. Spatial restraints resolve the ambiguity arising from interpretations of mutagenesis data

A structural model of pentameric phospholamban (Plb) in a lipid bilayer has been derived using a combination of experimental data, obtained from ATR-FTIR site-directed dichroism, and the implementation of the resulting restraints during a molecular dynamics simulation. Plb (residues 24-52) has been synthesised incorporating a new label, 1-(13)C==(18)O, at residues 42 and 43. We have not only determined the tilt of the helices, 10(+/-6) degrees, but also the relative orientation of the transmembrane segments, with an omega angle of -32(+/-10) degrees for L42. This angle is taken as zero in the direction of the helix tilt. Plb is a simple test case where site-directed dichroism has been applied to resolve the indeterminacy arising from the mutagenesis data available. The results presented point specifically to a single structural model for Plb.     

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm^-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm^-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.     

Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy

The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. Both M2 segments form straight transmembrane alpha-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular. A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.     

Structure and dynamics of membrane-associated ICP47, a viral inhibitor of the MHC I antigen-processing machinery

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.