Three Dimensional Structure of Mammalian Tachykinin Peptide Neurokinin B Bound to Lipid Micelles

Abstract

Neurokinin B (NKB), a decapeptide of mammalian origin exhibits a variety of biological activities such as regulatory functions in reproduction, pre-eclampsia and neuroprotection in Alzheimer's disease. In order to gain insight into structure-function relationship, three- dimensional structure of NKB has been investigated using CD spectropolarimetry and two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR) spectroscopy in aqueous and membrane mimetic solvents. Unambiguous NMR assignments of resonances have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and Nuclear Overhauser Effect Spectroscopy (NOESY) experiments. Distance constraints obtained from the NMR data have been used to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Our data show that a helical structure is induced in NKB, in presence of perdeuterated dodecyl phosphocholine (DPC) micelles, a membrane model system. Further, the conformation adopted by NKB in presence of DPC micelles represents a structural motif typical of neurokinin-3 selective agonists.     

Three-dimensional structure of the mammalian tachykinin peptide neurokinin A bound to lipid micelles

The solution structure of NKA, a decapeptide of mammalian origin, has been characterized by CD spectropolarimetry and 2D proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy in both aqueous and membrane mimetic solvents. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NKA prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region (D4-M10) of the peptide in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system. Though less defined the N-terminus also displays some degree of order and a possible turn structure. The conformation adopted by NKA in the presence of DPC micelles represents a structural motif typical of neurokinin-2 selective agonists and is similar to that reported for eledoisin in hydrophobic environment.     

Lipid induced conformation of the tachykinin peptide Kassinin

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.     

Protein Folding: A Few Random Thoughts

Abstract

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.     

Structure of outer membrane protein A transmembrane domain by NMR spectroscopy

We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.     

Determination of the membrane contact residues and solution structure of the helix F/G loop of prostaglandin I2 synthase

From our topological arrangement model of prostaglandin I(2) synthase (PGIS) created by homology modeling and topology studies, we hypothesized that the helix F/G loop of PGIS contains a membrane contact region distinct from the N-terminal membrane anchor domain. To provide direct experimental data we have explored the relationship between the endoplasmic reticulum (ER) membrane and the PGIS F/G loop using a constrained synthetic peptide to mimic PGIS residues 208-230 cyclized on both ends through a disulfide bond with added Cys residues. The solution structure and the residues important for membrane contact of the constrained PGIS F/G loop peptide were investigated by high-resolution 1H two-dimensional nuclear magnetic resonance (2D NMR) experiments and a spin label incorporation technique. Through the combination of 2D NMR experiments in the presence of dodecylphosphocholine (DPC) micelles used to mimic the membrane environment, complete 1H NMR assignments of the F/G loop segment have been obtained and the solution structure of the peptide has been determined. The PGIS F/G loop segment shows a defined helix turn helix conformation, which is similar to the three-dimensional crystallography structure of P450BM3 in the corresponding region. The orientation and the residues contacted with the membrane of the PGIS F/G loop were evaluated from the effect of incorporation of a spin-labeled 12-doxylstearate into the DPC micelles with the peptide. Three residues in the peptide corresponding to the PGIS residues L217 (L11), L222 (L16), and V224 (V18) have been demonstrated to contact the DPC micelles, which implies that the residues are involved in contact with the ER membrane in the native membrane-bound PGIS. These results provided the first experimental evidence to localize the membrane contact residues in the F/G loop region of microsomal P450 and are valuable to further define and understand the membrane topology of PGIS and those of other microsomal P450s in the native membrane environment.     

Solution structures and model membrane interactions of Ctriporin,an anti‐methicillin‐resistant Staphylococcus aureus Peptide from Scorpion Venom

Ctriporin peptide (Ctr), a novel antimicrobial peptide isolated from the venom of the scorpion Chaerilus tricostatus, shows a broad‐spectrum of antimicrobial activity and is able to inhibit antibiotic resistant pathogens, including Methicillin resistant Staphylococcus aureus, Methicillin Resistant Coagulase‐negative Staphylococcus, and Penicillin Resistant Staphylococcus epidermidis strains. To understand the active conformation of the Ctr peptide in membranes, we have investigated the interaction of Ctr with the negatively charged and zwitterionic membrane‐mimetic micelles such as sodium dodecyl sulphate (SDS) and n‐dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of Ctr interacted with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in Ctr. Moreover, we have determined the solution structures of Ctr in SDS and DPC micelles using nuclear magnetic resonance (NMR) spectroscopy. The structural comparison of Ctr in the presence of SDS and DPC micelles showed significant conformational changes. The observed structural differences of Ctr in anionic versus zwitterionic membrane‐mimetic micelles suggest that the mode of interaction of this peptide may be different in two environments which may account for its ability to differentiate bacterial and eukaryotic cell membrane. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1143–1153, 2014.     

Micelle bound structure and DNA interaction of brevinin‐2‐related peptide,an antimicrobial peptide derived from frog skin

Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Three-dimensional structure of neuropeptide k bound to dodecylphosphocholine micelles

Neuropeptide K (NPK), an N-terminally extended form of neurokinin A (NKA), represents the most potent and longest lasting vasodepressor and cardiomodulatory tachykinin reported thus far. NPK has been shown to have high selectivity for the NK2 receptor. Because the micelle-associated structure may be relevant to the NPK-receptor interaction, the three-dimensional structure of the NPK in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The interproton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment NPK lacks a definite secondary structure, although some turn-like elements are present in the N terminus. The structure is well-defined in the presence of dodecylphosphocholine micelles. The global fold of NPK bound to DPC micelles consists of two well-defined helices from residues 9 to 18 and residues 27 to 33 connected by a noncanonical beta turn. The N terminus of the peptide is characterized by a 3(10) helix or a series of dynamic beta turns. The conformational range of the peptide revealed by NMR and circular dichroism (CD) studies has been analyzed in terms of characteristic secondary features. The observed conformational features have been further compared to a NKA and neuropeptide gamma (NPgamma) potent endogenous agonist for the NK2 receptor.     

Structural Characterization of Neurokinin-3 Receptor Selective Peptide Agonist Scyliorhinin II Bound to DPC Micelles

Abstract

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12–18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.     

Structural characterization of neurokinin-3 receptor selective peptide agonist scyliorhinin II bound to DPC micelles

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12-18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.     

Structure and dynamics of the conserved protein GPI anchor core inserted into detergent micelles

A suitable approach which combines nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have been used to study the structure and the dynamics of the glycosylphosphatidylinositol (GPI) anchor Manalphal-2Manalpha1-6Manalphal -4GlcNalpha1-6myo-inositol-1-OPO(3)-sn-1,2-dimyristoylglycerol (1) incorporated into dodecylphosphatidylcholine (DPC) micelles. The results have been compared to those previously obtained for the products obtainable from (1) after phospholipase cleavage, in aqueous solution. Relaxation and diffusion NMR experiments were used to establish the formation of stable aggregates and the insertion of (1) into the micelles. MD calculations were performed including explicit water, sodium and chloride ions and using the Particle Mesh Ewald approach for the evaluation of the electrostatic energy term. The MD predicted three dimensional structure and dynamics were substantiated by nuclear overhauser effect (NOE) measurements and relaxation data. The pseudopentasaccharide structure, which was not affected by incorporation of (1) into the micelle, showed a complex dynamic behaviour with a faster relative motion at the terminal mannopyranose unit and decreased mobility close to the micelle. This motion may be better described as an oscillation relative to the membrane rather than a folding event.     

The interactions of the HIV gp41 fusion peptides with zwitterionic membrane mimics determined by NMR spectroscopy

The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an alpha helical conformation between the Ile4 to Leu12 or to Ala15 region. Simulated annealing showed that the helical region extends from Ile4 to Met19. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies.     

Model membrane interaction and DNA-binding of antimicrobial peptide Lasioglossin II derived from bee venom

Lasioglossins, a new family of antimicrobial peptide, have been shown to have strong antimicrobial activity with low haemo-lytic and mast cell degranulation activity, and exhibit cytotoxic activity against various cancer cells in vitro. In order to understand the active conformation of these pentadecapeptides in membranes, we have studied the interaction of Lasioglossin II (LL-II), one of the members of Lasioglossins family with membrane mimetic micelle Dodecylphosphocholine (DPC) by fluorescence, Circular Dichroism (CD) and two dimensional (2D) ¹H NMR spectroscopy. Fluorescence experiments provide evidence of interaction of the N-terminal tryptophan residue of LL-II with the hydrophobic core of DPC micelle. CD results show an extended chain conformation of LL-II in water which is converted to a partial helical conformation in the presence of DPC micelle. Moreover we have determined the first three-dimensional NMR structure of LL-II bound to DPC micelle with rmsd of 0.36 Å. The solution structure of LL-II shows hydrophobic and hydrophilic core formation in peptide pointing towards different direction in the presence of DPC. This amphipathic structure may allow this peptide to penetrate deeply into the interfacial region of negatively charged membranes and leading to local membrane destabilization. Further we have elucidated the DNA binding ability of LL-II by agarose gel retardation and fluorescence quenching experiments.     

pH-Dependent conformational changes and topology of a herpesvirus translocating peptide in a membrane-mimetic environment

Pol peptide, an oligopeptide corresponding to the 27 C-terminal amino acids of DNA polymerase from herpes simplex virus type 1, has recently been suggested to translocate from endosomal compartments into the cytosol after being intracellularly delivered via a protein carrier. While an acidic environment was thought to be important for Pol peptide membrane translocation, the mechanism of translocation remains unclear. To investigate the influence of an acidic environment on the conformational properties of the peptide and on its propensity to interact with lipid bilayers, we characterized the structure of Pol peptide at different pH values by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The influence of detergent micelles, which mimic biological lipid membranes, on the peptide secondary structure was also studied. Our CD results indicate that the peptide is in a random conformation in aqueous solution at both acidic and basic pH, whereas in the presence of dodecylphosphocholine (DPC) micelles, it assumes a partial alpha-helical structure which is significantly pH-dependent. An NMR study confirmed that, in the presence of DPC micelles, a short C-terminal alpha-helix is present at pH 6.5, whereas almost two-thirds of the peptide (residues 10-26) fold into an extended amphipathic alpha-helix at pH 4.0. The orientation of Pol peptide relative to the DPC micelle was investigated using paramagnetic probes at both pH 4.0 and 6.5. These studies show that the peptide inserts deeply into the micelle at pH 4.0, whereas it is more exposed to the aqueous environment at pH 6.5. On the basis of these results, a model which might explain the mechanism of translocation of Pol peptide from acidic endosomes to the cytosol is discussed.     

Interactions between mastoparan B and the membrane studied by 1H NMR spectroscopy

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic alpha-helical structure from Lys2 to Leu14. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp9 are in close contact with methylene protons of SDS micelles. T1 relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp9 with Ala9, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp9 into the membrane, as well as the amphiphilic alpha-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

Structure of epidermal growth factor bound to perdeuterated dodecylphosphocholine micelles determined by two-dimensional NMR and simulated annealing calculations.

The interaction of mouse epidermal growth factor (mEGF) with micelles of a phospholipid analogue, perdeuterated dodecylphosphocholine (DPC), was investigated by two-dimensional 1H NMR. Sequence-specific resonance assignments of the micelle-bound mEGF have been made, and the chemical shifts were compared with those in the absence of DPC. DPC induced large chemical shift changes of the resonances from the residues in the C-terminal tail (residues 46-53) but little perturbation on the residues in the main core (residues 1-45). Starting from the three-dimensional structure in the absence of DPC, micelle-bound structures were calculated using the program XPLOR with interproton distance data obtained from NOESY spectra recorded in the presence of DPC. The C-terminal tail of mEGF was found to change conformation to form an amphiphilic structure when bound to the micelles. It is possible that induced fit in the C-terminal tail of mEGF occurs upon binding to a putative hydrophobic pocket of the EGF receptor.     

Role of the helical structure of the N-terminal region of Plasmodium falciparum merozoite surface protein 2 in fibril formation and membrane interaction

Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP2(1-25)) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP2(1-25) in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP2(1-25) was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP2(1-25) was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP2(1-25) in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10-24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP2(1-25) fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.     

The interactions of the HIV gp41 fusion peptides with zwitterionic membrane mimics determined by NMR spectroscopy

The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an α helical conformation between the Ile⁴ to Leu¹² or to Ala¹⁵ region. Simulated annealing showed that the helical region extends from Ile⁴ to Met¹⁹. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies.     

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles.

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.