1H-nmr studies on the structure of a new thionin from barley endosperm

A new thionin from barley, ω-hordothionin, has been shown to exist in aqueous solution as a mixture of two different isoforms in a 3:2 ratio, as revealed by a complete analysis of its two-dimensional ¹H-nmr spectra. The conformational heterogeneity arises frtm cis–trans isomerism ahout the Phe 12–Pro 13 peptide bond, where the major, form corresponds to the cis conformation. The complete assignment of chemical shifts and nuclear Overhaiiser effects (NOES) of the two isoforms allow a detailed comparative analysis of their conformational properties, even though a complete calculation of their solution structures is not possible because of a somewhat limited number of NOE constraints. Structures for the two isomers could be modeled, however, on the basis of the high structural homology between ω-hordothionin and related γ-thionins, and under the conditions of satisfying all observed experimental data. The two isoforms adopt practically identical global folds and the structural changes imposed by cis–trans isomerization are confined to the region proximal to Pro 13. The cis–trans isomerism occurs in a conserved loop connecting the first β-strand of the triple-stranded antiparallel β-sheet and the α-helix. A comparative analysis of the sequences of this loop in the different thionins suggests that the cis–trans equilibrium about the X-Pro peptide bond depends on the size of the side chain of X (X = Gly in γ-thionins and Phe in ω-thionin). The structural homology of this new thionin with γ-thionins as well as with some scorpion toxins and insect defensins suggests that these proteins may share a common mode of functional activity. © 1995 John Wiley & Sons, Inc.     

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.     

Evidence for spontaneous segregation phenomena in mixed micelles of gangliosides

A light scattering study of the effect of mixing in aqueous solution two gangliosides, GM2 and GT1b, having different hydrophilic headgroups and similar lipid moieties is presented. Mixed micelle formation with spatial segregation of one ganglioside with respect to the other was observed. It is also shown that segregation is a spontaneous phenomenon which is explainable only in terms of simple geometrical arguments, that is by the fact that the large headgroup of GT1b provides the lipidic core of the aggregate with a better shielding from water in the highly curved regions than the smaller headgroup of GM2 can do. This finding may be of help in understanding the behaviour of gangliosides in artificial and natural membranes.     

Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.     

One- and two-dimensional NMR relaxation studies of dynamics and structure in bile salt-phosphatidylcholine mixed micelles

A series of one- and two-dimensional 1H-NMR relaxation measurements has been conducted on simple and mixed micellar aggregates of taurocholate, diphenylvaleroylphosphatidylcholine (diPVPC) and egg yolk phosphatidylcholine (egg PC). The results are interpreted to provide structural and dynamic comparisons between micelles and vesicles, between phospholipids of varying chain length, and between different lipid components within the same micellar aggregate. Both chemical shift changes and two-dimensional nuclear Overhauser effect cross peaks suggest direct interaction of taurocholate and PC chemical sites, although the latter observations may also be accounted for by PC-PC interactions. These experiments demonstrate the promise of NMR relaxation techniques for investigations of molecular organization in model substrate for lipolytic enzymes.     

Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues

Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H⁺-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H⁺-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185–97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (λ_max), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k_q) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We tentatively assigned the two major Trp lifetimes to the Trp (χ₁=60° and 180°) rotamers, based on the recent lifetime–rotamer correlation proposed for model cyclic peptides (Pan and Barkley 2004, Biophys J 86:3828–35). We also analyzed the role of the hydrophobic anchor, by comparing the micelle binding of fragments of various lengths including the synthesized full-length protein and detected peculiar differences for protein interaction with the polar headgroups of DM or DPC.     

Evidence for a C-terminal structural motif in gastrin and its bioactive fragments in membrane mimetic media

The conformational preferences of human little gastrin, [Nle(15)] gastrin-17, and its short analogues, gastrin-4 and [beta-Ala(1)] gastrin-5, which include the C-terminal tetrapeptide sequence Trp-Met-Asp-Phe-NH(2) crucial for gastrin bioactivity, were determined by NMR spectroscopy in aqueous solutions of zwitterionic dodecylphosphocholine micelles. Backbone HN chemical shift temperature variance, Halpha chemical shift deviations and complex non-sequential NOE patterns pointed to the C-terminal of [Nle(15)] gastrin-17 adopting an ordered conformation. Distance geometry calculations and NOE-restrained molecular dynamics simulations in membrane mimetic solvent boxes of decane and water indicated the C-terminal tetrapeptide sequence of all three peptides adopted a similar, well defined structure, with a general type IV beta-turn observed for all three peptides. The conformation of [Nle(15)] gastrin-17 consisted of two short helices between Leu(5)-Glu(9) and Ala(11)-Trp(14), with the one helix terminating in a type I beta-turn spanning Gly(13)-Asp(16). The experimental evidence and conformational characteristics of the three peptides in micellar media support a membrane-associated mechanism of receptor recognition and activation for the gastrin hormone family and furthermore point to a possible biologically relevant structural motif for gastrin activity.     

500 MHz 1H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

Caveolin-1 hydrophobic segment peptides insertion into membrane mimetic systems: role of proline residue

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122 that are anchored to the membrane with cholesterol-rich domains. Previously, we reported that changing the Pro(110) residue to Ala (the P110A mutant) prevents not only the localization of the protein into lipid rafts but also the formation and functioning of caveolae. The conformational state of caveolin-1 can be shifted toward the transmembrane arrangement by this single amino acid mutation. To model the conformation, and extent of membrane insertion of this segment into membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of caveolin-1 having the sequence KKKKLSTIFGIPMALIWGIYFAILKKKKK-amide and the mutated version, KKKKLSTIFGIAMALIWGIYFAILKKKKK-amide. These peptides contain flanking Lys residues to facilitate purification and handling of the peptide. Circular dichroism measurements demonstrated that the mutated peptide has increased helical content compared with the wild type both in the presence and absence of lipid. The fluorescence emission from the Trp residues in the peptide showed significant blue shifts in the presence of liposomes, however the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. Our overall findings support our studies with the intact protein in cells and suggest that the peptide of WT caveolin-1 hydrophobic segment has an intrinsic preference not to maintain its conformation as a rigid transmembrane helix. Substituting the Pro residue with an Ala allows the peptide to exist in a more hydrophobic environment likely as a consequence of a change in its conformation to a straight hydrophobic helix that traverses the membrane.     

NMR structural comparison of the cytoplasmic juxtamembrane domains of G-protein-coupled CB1 and CB2 receptors in membrane mimetic dodecylphosphocholine micelles

The fourth cytoplasmic domain, the so-called C-terminal juxtamembrane segment or helix VIII, has been identified in numerous G-protein-coupled receptors and exhibits unique functional characteristics. Efforts have been devoted to studying the juxtamembrane segment in order to understand the biological importance of the segment in G-protein activation of the cannabinoid CB1 and CB2 receptors. Recent biochemical data revealed that the CB1 C-terminal juxtamembrane peptide fragment CB1-(401-417) can directly activate the G-protein and also showed that the specificity of the signal transduction activation by the C-terminal juxtamembrane region is unique to the CB1 receptor but not to the CB2 receptor (Mukhopadhyay, S., and Howlett, A. C. (2001) Eur. J. Biochem. 268, 499-505). However, there is experimental work, not yet reported, on the conformational analyses and structural comparison between the respective helix VIII segments of the two receptors. In the present study, we have examined the conformational specificities of the cytoplasmic helical domains for both cannabinoid receptors. Three-dimensional structural features of two synthetic CB1 and CB2 peptides, CB1I397-G418 and CB2I298-K319, respectively, in membrane mimetic DPC micelles were studied using a combined high resolution NMR and computer modeling approach. Comparisons of the NMR-determined structures of the two peptides as well as their correspondent mutant peptides revealed their conformational properties and salt bridge dissimilarity, which might help us to understand the different structural roles of the fourth cytoplasmic helices in the function and regulation of CB1 and CB2 receptors.     

Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein

Measuring the stability of integrated membrane proteins under equilibrium conditions is hampered by the nature of the proteins' amphiphilic environment. While intrinsic fluorescence is a useful probe for structural changes in water-soluble proteins, the fluorescence of membrane proteins is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a kinetic analysis of the folding of a membrane protein, disulfide bond reducing protein B (DsbB), in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate (SDS) and dodecyl maltoside (DM). This analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. Refolding and unfolding occur on the second to millisecond timescale and involve only one relaxation phase, when monitored by conventional stopped-flow. The kinetic data indicate that denaturation occurs around 0.3 mole fraction of SDS, in agreement with CD analysis and acrylamide quenching data. The rate constants have been fit to a three-state folding scheme involving the SDS-denatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of SDS. The stability of DsbB is around 4.4 kcal/mol in DM, and this is halved upon reduction of the two periplasmic disulfide bonds, and is sensitive to mutagenesis. With the caveat that kinetic data are always open to alternative interpretations, time-resolved studies in mixed micelles provide a useful approach to measure membrane protein stability over a wide range of concentrations of SDS and DM, as well as a framework for the future characterization of the DsbB folding mechanism.     

Solution three-dimensional structure of surfactin: A cyclic lipopeptide studied by 1H-nmr,distance geometry,and molecular dynamics

The solution three-dimensional structure of the protonated [Leu7]-surfactin, an hepta-peptide extracted from Bacillus subtilis, has been determined from two-dimensional ¹Hnmr performed in ²H₆-dimethylsulfoxide and combined with molecular modeling. Experimental data included 9 coupling constants, 61 nuclear Overhauser effect derived distances, NH temperature coefficients, and ¹³C relaxation times. Two distance geometry (DISMAN) protocols converged toward models of the structure and the best of them were refined by restrained and unrestrained molecular dynamics (GROMOS). Two structures in accord with the set of experimental constraints are presented. Both are characterized by a “horse saddle” topology for ring atoms on which are attached the two polar Glu and Asp side chains showing an orientation clearly opposite to that of the C_11–13 aliphatic chain. Amphipathic and surface properties of surfactin are certainly related to the existence of such minor polar and a major hydrophobic domains. The particular “claw” configuration of acidic residues observed in surfactin gives important clues for the understanding of its cation binding and transporting ability. © 1994 John Wiley & Sons, Inc.     

Evidence for a metalloprotein structure of plasma membrane 5'-nucleotidase

To point out the metalloprotein structure of bovine liver plasma membrane 5'-nucleotidase, we studied the inhibition mechanism of the purified enzyme by EDTA: this apparently non-competitive inhibition seems to be dependent on EDTA concentration, pH, temperature and incubation time. When the restoration of activity was assayed by addition of divalent cations or by gel filtration, the inhibition became progressively irreversible with time. Incubation of the enzyme with [14C]EDTA allowed us to observe, after gel filtration as well as after sucrose gradient ultracentrifugation, that the chelating agent is bound to 5'-nucleotidase.     

500 MHz H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

1H-nmr comparative studies of polynucleotides: Conformation and dynamic structure of polyribo(uridylic) and polyribo(cytidylic) acids in neutral solution

The conformation and the dynamic structure of single-stranded poly(U) and poly(C) in neutral aqueous solution have been investigated by ¹H-nmr at two different frequencies (90 and 250 MHz) and at various temperatures. Measurements of proton chemical shifts, coupling constants J_H-H, and proton relaxation times, T₁, T₂, versus temperature show a striking difference in conformation and in dynamic structure between the two polynucleotides studied. The temperature effect on δ and J_H-H is found to be substantial for poly(C) and insignificant for poly(U). The S conformer is favored in poly(U), whereas the N conformer strongly predominates in poly(C) (?90%), similar to the case for RNAs. These results suggest that single-stranded poly(C) probably possesses a helical or partial helical structure, whereas poly(U) shows a clear preference for the random coil, in agreement with the optical results. The local motions of the ribose and base were studied at various temperatures by measurements on the relaxation times at 90 and 250 MHz. For a given temperature between 22 and 72°C, the ratio T₁(90)/T₁(250) is practically the same for all poly(U) protons, indicating that in this temperature interval the ribose base unit of poly(U) undergoes an isotropic motion characterized by a single correlation time τ_c. Above 52°C, poly(C) exhibits a dynamic structure similar to poly(U). Below this temperature, poly(C) exists in an equilibrium between randomly coiled and single-stranded helix forms. This situation is characterized by a strong cross-relaxation effect and T₁ values corresponding to a relatively short apparent correlation time. An activation energy of 4 kcal/mol was determined for the motion of the ribose–base unit in both single-stranded polynucleotides.     

Isotropic solutions of phospholipid bicelles: A new membrane mimetic for high-resolution NMR studies of polypeptides

In order to illustrate the utility of phospholipid bicelles [Sanders, C.R. and Schwonek, J.P.(1992) Biochemistry, 31, 8898–8905] as a membrane mimetic for high-resolution NMRstudies, we have recorded two-dimensional 1H NMR spectra of the tetradecameric peptidemastoparan Vespula lewisii in an isotropic aqueous solution of dimyristoyl and dihexanoylphosphatidylcholine. Mastoparan is largely unstructured in water, but assumes a well-definedhelical conformation in association with the bilayers. A pronounced periodicity of thesequential NH chemical shifts provides strong evidence that the helix axis of this shortpeptide is parallel, rather than perpendicular, to the bilayer plane. The bicellar solutions stillrequire in-depth morphological characterization, but they appear to be ideal media for NMRdetermination of the mode of binding and the structure of membrane-associated peptides andproteins.     

Proton magnetic resonance relaxation studies on the structure of mixed micelles of Triton X-100 and dimyristoylphosphatidylcholine.

Proton magnetic resonance and gel chromatographic studies on mixtures of phospholipid and the nonionic surfactant Triton X-200 have shown that at temperatures above the thermotropic phase transition of the phospholipid and below the cloud point of Triton, mixed micelles are present at molar ratios above about 2:1 Triton/phospholipid. Proton T1 and T2 (from line widths) relaxation times are reported for protons in Triton micelles and in mixed micelles of Triton and dimyristoylphosphatidylcholine at a molar ratio of 3:1 Triton/phospholipid. The T1 values and their temperature dependence and the activation energies of the various Triton proton groups appear to reflect internal motions of the Triton molecules in the micelle. Measurements of the T1/T2 ratio and frequency dependence (55-220 MHz) suggest that the hydrophobic tert-butyl group in Triton is observed under extreme narrowing conditions. The T1 and T2 values of Triton are unchanged in the presence of phosphatidylcholine. The T1 values of various protons of dimyristoylphosphatidylcholine in mixed micelles are similar to those reported for the phospholipid in sonicated vesicles, which are used as membrane models, and presumably the same coupled trans-gauche motions dominate. The T2 values for the terminal methyl and choline methyl protons in the phospholipid are longer than those reported for these groups in vesicles. Hence, the motion of the phospholipid in the mixed micelles appears to be less restricted than in vesicles. T1 measurements in H20/D20 mixtures are consistent with the idea that water does not penetrate the hydrophobic core of the mixed micelles, while water does solvate the polar oxyethylene and choline methyl groups. Titration with Mn2+ confirms that the oxyethylene and choline methyl groups are on the exterior of the mixed micelle while the hydrophobic groups are located in the micellar interior.