Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution

Melittin is a cationic, amphipathic, hemolytic peptide composed of 26 amino acid residues. It is intrinsically fluorescent due to the presence of a single tryptophan residue, which has been shown to be crucial for its hemolytic activity. It undergoes a structural transition from a random coil monomer to an alpha-helical tetramer at high ionic strength. Although the aggregation behavior of melittin in solution is well characterized, dynamic information associated with the aggregation of melittin is lacking. In this paper, we have monitored the effect of ionic strength on the dynamics and aggregation behavior of melittin in aqueous solution by utilizing sensitive fluorescence approaches, which include the red edge excitation shift (REES) approach. Importantly, we demonstrate that REES is sensitive to the self-association of melittin induced by ionic strength. The change in environment experienced by melittin tryptophan(s) is supported by changes in fluorescence emission maximum, polarization, and lifetime. In addition, the accessibility of the tryptophan residue was probed by fluorescence quenching experiments using acrylamide and trichloroethanol as soluble and hydrophobic quenchers, respectively. Circular dichroism studies confirm the ionic strength-induced change in the secondary structure of melittin. Taken together, these results constitute the first report showing that REES could be used as a sensitive tool to monitor the aggregation behavior of melittin in particular and other proteins and peptides in general.     

Orientation and rotational dynamics of spin-labeled phalloidin bound to actin in muscle fibers

We have used electron paramagnetic resonance spectroscopy (EPR) to investigate the orientational distribution of actin in thin filaments of glycerinated muscle fibers in rigor, relaxation, and contraction. A spin-labeled derivative of a mushroom toxin, phalloidin (PHSL), was bound to actin in the muscle fibers (PHSL–fibers). The EPR spectrum of unoriented PHSL–labeled myofibrils consisted of three sharp lines with a splitting between the outer extrema (2T) of 42.8 ± 0.1 G, indicating that the spin labels undergo restricted nanosecond rotational motion within an estimated halfcone angle of 76°. When the PHSL–fiber bundle was oriented parallel to the magnetic field, the splitting between the zero-crossing points (2T′) was 42.7 ± 0.1 G. When the fiber bundle was perpendicular to the magnetic field, 2T′ decreased to 34.5 ± 0.2 G. This anisotropy shows that the motion of the probe is restricted in orientation by its binding site on actin, so that the EPR spectrum of PHSL–fiber bundles would be sensitive to small changes in the mean axial orientation of the PHSL–actin interface. No differences in the EPR spectra were observed in fibers during rigor, relaxation, or contraction, indicating that the mean axial orientation of the PHSL binding site changes by less than 5°, and that the amplitude of nanosecond probe rotational motion, which should be quite sensitive to the local environment of the phalloidin, changes by no more than 1°. These results rule out large changes in the overall geometry of the actin filament and in the local conformation of actin near the phalloidin binding site during the generation of isometric tension in muscle fibers. © 1993 Wiley-Liss, Inc.     

The dynamic properties of melittin in solution

Molecular dynamics simulations are described for the peptide melittin. The atomic trajectories are calculated both with normal potential energy functions and with additional distance restraints deduced from nuclear Overhauser effects observed in NMR experiments. The results are compared with NRM data on coupling constants and amide exchange rates and witt B-factors from X-ray crystallography. The observed correlations between experiment and molecular dynamics simulations suggest a relatively mobile C-terminus and relatively high flexibility around residue 11. It is noted that the high conformational variation around residue 11 is due in part to the presence of a proline at position 14 which results in a missing H-bond in the largely -helical structure. It is also noted that a proline is a common feature of many putative membrane spanning helices. A role for such prolines is suggested.     

Synaptotagmin 1 and SNAREs form a complex that is structurally heterogeneous

Synaptotagmin 1 (syt1) functions as a Ca²⁺-sensor for neuronal exocytosis. Here, site-directed spin labeling was used to examine the complex formed between a soluble fragment of syt1, which contains its two C2 domains, and the neuronal core soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Changes in electron paramagnetic resonance lineshape and accessibility for spin-labeled syt1 mutants indicate that in solution, the assembled core SNARE complex contacts syt1 in several regions. For the C2B domain, contact occurs in the polybasic face and sites opposite the Ca²⁺-binding loops. For the C2A domain, contact is seen with the SNARE complex in a region near loop 2. Double electron-electron resonance was used to estimate distances between the two C2 domains of syt1. These distances have broad distributions in solution, which do not significantly change when syt1 is fully associated with the core SNARE complex. The broad distance distributions indicate that syt1 is structurally heterogeneous when bound to the SNAREs and does not assume a well-defined structure. Simulated annealing using electron paramagnetic resonance-derived distance restraints produces a family of syt1 structures where the Ca²⁺-binding regions of each domain face in roughly opposite directions. The results suggest that when associated with the SNAREs, syt1 is configured to bind opposing bilayers, but that the syt1/SNARE complex samples multiple conformational states.     

Conformational analysis of melittin in solution phase: vibrational circular dichroism study

The vibrational absorption and vibrational circular dichroism (VCD) spectra of melittin in D(2)O solutions at different pH values, different salt concentrations, or different 2,2,2-trifluoroethanol (TFE) concentrations are recorded in the amide I' (1850-1600 cm(-1)) region. Two models are used to simulate this peptide in different conditions, and a coupled oscillator program is used to obtain the calculated absorption and VCD spectra. This study indicates that melittin adopts a mixed structure in D(2)O solution at low pH, low salt concentration, or low TFE concentration. With an increase in pH, salt concentration, or TFE concentration, the structure changes to alpha-helix and further increases lead to aggregation. These results demonstrate the versatility of VCD in probing the conformations of peptides under different environmental perturbations.     

Direct evidence that the carboxyl-terminal sequence of a bacterial chemoreceptor is an unstructured linker and enzyme tether

Sensory adaptation in bacterial chemotaxis involves reversible methylation of specific glutamyl residues on chemoreceptors. The reactions are catalyzed by a dedicated methyltransferase and dedicated methylesterase. In Escherichia coli and related organisms, control of these enzymes includes an evolutionarily recent addition of interaction with a pentapeptide activator located at the carboxyl terminus of the receptor polypeptide chain. Effective enzyme activation requires not only the pentapeptide but also a segment of the receptor polypeptide chain between that sequence and the coiled-coil body of the chemoreceptor. This segment has features consistent with a role as a flexible and presumably unstructured linker and enzyme tether, but there has been no direct information about its structure. We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to characterize structural features of the carboxyl-terminal 40 residues of E. coli chemoreceptor Tar. Beginning ～ 35 residues from the carboxyl terminus and continuing to the end of the protein, spectra of spin-labeled Tar embedded in native membranes or in reconstituted proteoliposomes, exhibited mobilities characteristic of unstructured, disordered segments. Binding of methyltransferase substantially reduced mobility for positions in or near the pentapeptide but mobility for the linker sequence remained high, being only modestly reduced in a gradient of decreasing effects for 10-15 residues, a pattern consistent with the linker providing a flexible arm that would allow enzyme diffusion within defined limits. Thus, our data identify that the carboxyl-terminal linker between the receptor body and the pentapeptide is an unstructured, disordered segment that can serve as a flexible arm and enzyme tether.     

Structural determinants of nitroxide motion in spin-labeled proteins: tertiary contact and solvent-inaccessible sites in helix G of T4 lysozyme

A nitroxide side chain (R1) has been substituted at single sites along a helix-turn-helix motif in T4 lysozyme (residues 114-135). Together with previously published data, the new sites reported complete a continuous scan through the motif. Mutants with R1 at sites 115 and 118 were selected for crystallographic analysis to identify the structural origins of the corresponding two-component EPR spectra. At 115, R1 is shown to occupy two rotamers in the room temperature crystal structure, one of which has not been previously reported. The two components in the EPR spectrum apparently arise from differential interactions of the two rotamers with the surrounding structure, the most important of which is a hydrophobic interaction of the nitroxide ring. Interestingly, the crystal structure at 100 K reveals a single rotamer, emphasizing the possibility of rotamer selection in low-temperature crystal structures. Residue 118 is at a solvent-inaccessible site in the protein core, and the structure of 118R1, the first reported for the R1 side chain at a buried site, reveals how the side chain is accommodated in an overpacked core.     

Structure of bacteriocin AS-48: from soluble state to membrane bound state

The bacteriocin AS-48 is a membrane-interacting peptide, which displays a broad anti-microbial spectrum against Gram-positive and Gram-negative bacteria. The NMR structure of AS-48 at pH 3 has been solved. The analysis of this structure suggests that the mechanism of AS-48 anti-bacterial activity involves the accumulation of positively charged molecules at the membrane surface leading to a disruption of the membrane potential. Here, we report the high-resolution crystal structure of AS-48 and sedimentation equilibrium experiments showing that this bacteriocin is able to adopt different oligomeric structures according to the physicochemical environment. The analysis of these structures suggests a mechanism for molecular function of AS-48 involving a transition from a water-soluble form to a membrane-bound state upon membrane binding.     

Collisions between helical peptides in membranes monitored using electron paramagnetic resonance: evidence that alamethicin is monomeric in the absence of a membrane potential.

Alamethicin is a 20-amino-acid peptide that produces a voltage-dependent conductance in membranes. We investigated the state of aggregation of alamethicin in egg phosphatidylcholine and dioleoylphosphatidylcholine membranes by examining the EPR spectra obtained from an active analog of this peptide that is spin-labeled at its C-terminus. The dependence of both the linewidth and signal intensity as a function of peptide concentration exhibit exchange broadening as the peptide concentration is increased; however, the exchange rates are linear with peptide concentration as is expected for the simple diffusion of monomers. In addition, the spin-exchange rates obtained from the linebroadening are consistent with collisional rates that are predicted from free Brownian diffusion. The results provide strong evidence that in the absence of a membrane potential, alamethicin is largely monomeric in these membranes.     

Combined NMR and EPR spectroscopy to determine structures of viral fusion domains in membranes

Methods are described to determine the structures of viral membrane fusion domains in detergent micelles by NMR and in lipid bilayers by site-directed spin labeling and EPR spectroscopy. Since in favorable cases, the lower-resolution spin label data obtained in lipid bilayers fully support the higher-resolution structures obtained by solution NMR, it is possible to graft the NMR structural coordinates into membranes using the EPR-derived distance restraints to the lipid bilayer. Electron paramagnetic dynamics and distance measurements in bilayers support conclusions drawn from NMR in detergent micelles. When these methods are applied to a structure determination of the influenza virus fusion domain and four point mutations with different functional phenotypes, it is evident that a fixed-angle boomerang structure with a glycine edge on the outside of the N-terminal arm is both necessary and sufficient to support membrane fusion. The human immunodeficiency virus fusion domain forms a straight helix with a flexible C-terminus. While EPR data for this fusion domain are not yet available, it is tentatively speculated that, because of its higher hydrophobicity, a critically tilted insertion may occur even in the absence of a kinked boomerang structure in this case.     

Application of percolation theory principles to the analysis of interaction of adenylate cyclase complex proteins in cell membranes

Summary Lateral protein movement in cell membranes takes place in a medium with obstacles. These obstacles are: (a) aggregates of major integral proteins immobilized by submembraneous structures and cytoskeleton, and (b) membrane lipids in the gel phase. Hormonal activation of the adenylate cyclase complex is associated with lateral mobility of the constituent proteins. Modification of the interaction of these proteins due to variation of the fluid lipid fraction in reticulocyte membranes has been studied. A decrease in the percentage of fluid lipids in membranes resulted in the inhibition (up to the full cessation) of the interaction of -adrenoreceptors with regulatory N_s-proteins. The interaction of N_s-proteins with catalytic proteins stopped as well. On the other hand, an increase in the fluid lipid fraction led to a more intensive interaction. These facts do not arise from the functional damage of interacting proteins. Conseqently, hormonal activation of the adenylate cyclase complex depends on the fraction of fluid lipids in the membrane. The data obtained are in conformity with the percolation theory which makes it possible to characterize long-distance protein movement in a medium (fluid lipids) containing obstacles. Thus, interacting proteins prove to diffuse within distances greatly exceeding protein sizes. As a consequence, the intrinsic activity of a -agonist, isoproterenol, varies from 1 to 0 depending on the fluid lipid fraction. Our findings also suggest that in vitro there are no -receptors precoupled with N_s-proteins in rat reticulocyte membranes in the absence of guanine nucleotides.     

The tryptophan switch: changing ligand-binding specificity from type I to type II in SH3 domains

The ability of certain Src homology 3 (SH3) domains to bind specifically both type I and type II polyproline ligands is perhaps the best characterized, but also the worst understood, example in the family of protein-interaction modules. A detailed analysis of the structural variations in SH3 domains, with respect to ligand-binding specificity, together with mutagenesis of SH3 Fyn tyrosine kinase, reveal the structural basis for types I and II binding specificity by SH3 domains. The conserved Trp in the SH3 binding pocket can adopt two different orientations that, in turn, determine the type of ligand (I or II) able to bind to the domain. The only exceptions are ligands with Leu at positions P(-1) and P(2), that deviate from standard poly-Pro angles. The motion of the conserved Trp depends on the presence of certain residues located in a key position (132 for Fyn), near the binding pocket. SH3 domains placing aromatic residues in this key position are promiscuous. By contrast, those presenting beta-branched or long aliphatic residues block the conserved Trp in one of the two possible orientations, preventing binding in a type I orientation. This is experimentally demonstrated by a single mutation in Fyn SH3 (Y132I) that abolishes type I ligand binding, while preserving binding to type II ligands. Thus, simple conformational changes, governed by simple rules, can have profound effects on protein-protein interactions, highlighting the importance of structural details to predict protein-protein interactions.     

A comparison of the dynamic behavior of monomeric and dimeric insulin shows structural rearrangements in the active monomer

Molecular dynamics (MD) simulations (5-10ns in length) and normal mode analyses were performed for the monomer and dimer of native porcine insulin in aqueous solution; both starting structures were obtained from an insulin hexamer. Several simulations were done to confirm that the results obtained are meaningful. The insulin dimer is very stable during the simulation and remains very close to the starting X-ray structure; the RMS fluctuations calculated from the MD simulation agree with the experimental B-factors. Correlated motions were found within each of the two monomers; they can be explained by persistent non-bonded interactions and disulfide bridges. The correlated motions between residues B24 and B26 of the two monomers are due to non-bonded interactions between the side-chains and backbone atoms. For the isolated monomer in solution, the A chain and the helix of the B chain are found to be stable during 5ns and 10ns MD simulations. However, the N-terminal and the C-terminal parts of the B chain are very flexible. The C-terminal part of the B chain moves away from the X-ray conformation after 0.5-2.5ns and exposes the N-terminal residues of the A chain that are thought to be important for the binding of insulin to its receptor. Our results thus support the hypothesis that, when monomeric insulin is released from the hexamer (or the dimer in our study), the C-terminal end of the monomer (residues B25-B30) is rearranged to allow binding to the insulin receptor. The greater flexibility of the C-terminal part of the beta chain in the B24 (Phe-->Gly) mutant is in accord with the NMR results. The details of the backbone and side-chain motions are presented. The transition between the starting conformation and the more dynamic structure of the monomers is characterized by displacements of the backbone of Phe B25 and Tyr B26; of these, Phe B25 has been implicated in insulin activation.     

Lipid-protein interactions: NMR study of melittin and its binding to lysophosphatidylcholine

Proton NMR of melittin differs according to the association state of the peptide in the monomer or tetramer. Melittin interacts with lysophosphatidylcholine micelles, whatever the association state of melittin; well resolved superimposed spectra from both components for all the lipid to peptide molar ratios are observed. Within the complexes, local mobility and fast exchange occurs. On binding concomitant shifts on Trp₁₉ indole lines and on the aliphatic CH₂ protons of the lipids are detected. The lipid perturbation is maximum for methylene groups in α and β of the ester bond, this could allow positionning of Trp₁₉ in the hydrophobic core of the lipids.     

Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy.

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.     

Peptide antibiotic trichogin in model membranes: Self-association and capture of fatty acids

The antimicrobial action of peptides in bacterial membranes is commonly related to their mode of self-assembling which results in pore formation. To optimize peptide antibiotic use for therapeutic purposes, a study on the concentration dependence of self-assembling process is thus desirable. In this work, we investigate this dependence for peptaibol trichogin GA IV (Tric) in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane in the range of peptide concentrations between 0.5 and 3.3?mol%. Pulsed double electron-electron resonance (PELDOR) applied on spin-labeled peptide analogs highlights the onset of peptide dimerization above a critical peptide concentration value, namely ~ 2?mol%. Electron spin echo (ESE) envelope modulation (ESEEM) for D₂O-hydrated bilayers shows that dimerization is accompanied by peptide re-orientation towards a trans-membrane disposition. For spin-labeled stearic acids (5-DSA) in POPC bilayers, the study of ESE decays and ESEEM in the presence of a deuterated peptide analog indicates that above the critical peptide concentration the 5-DSA molecules are attracted by peptide molecules, forming nanoclusters. As the 5-DSA molecules represent a model for the behavior of fatty acids participating in bacterial membrane homeostasis, such capturing action by Tric may represent an additional mechanism of its antibiotic activity.     

The interaction of phospholipid membranes with poly(ethylene glycol). Vesicle aggregation and lipid exchange

(1) The water soluble polymer, poly(ethylene glycol), causes aggregation of sonicated vesicles of dimyristoylphosphatidylcholine in a manner consistent with a steric exclusion mechanism. (2) Poly(ethylene glycol) promotes the exchange of lipids between multilamellar vesicles of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine when the lipids are in the liquid-crystalline state. (3) ³¹P-NMR studies demonstrate that the bilayer configuration of smectic mesophases of dipalmitoylphosphatidylcholine is substantially maintained in the presence of poly(ethylene glycol).     

Autoreduction and aggregation of fungal laccase in solution phase: possible correlation with a resting form of laccase

This paper reports results of a reexamination of some poorly understood peculiarities of laccases, an enzyme family which has been extensively studied in our laboratories as well as by others for some years. The issue that is reconsidered here is the previously proposed existence of "active" and "resting" forms of laccases. The presence of fungal laccases with partly reduced active sites is demonstrated. Of further interest is that an aggregated state in solution, not to our knowledge previously noted for laccase, has been found by using small-angle X-ray scattering as well as thorough analysis of the results of several biochemical experiments. Under some conditions, this aggregated state may correlate with the resting form of the laccases, although this resting form could have a broader significance. It was shown that Trametes ochracea laccase had some anomalous characteristics, which could be correlated with the high concentration of the "resting" enzyme. The mechanism of formation of resting laccase is suggested. Knowledge of the resting state is of importance for in vitro studies. Additionally, a suggestion about the possible regulatory role of this form in vivo is mentioned.     

The interaction of neuropeptide Y with negatively charged and zwitterionic phospholipid membranes

The interaction of the 36 amino acid neuropeptide Y (NPY) with liposomes was studied using the intrinsic tyrosine fluorescence of NPY and an NPY fragment comprising amino acids 18–36. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the experimentally measured binding curves, the standard Gibbs free energy for the peptide transfer from aqueous solution to the lipid membrane was calculated to be around ?30 kJ/mol for membrane mixtures containing physiological amounts of acidic lipids at pH 5. The effective charge of the peptide depends on the pH of the buffer and is about half of its theoretical net charge. The results were confirmed using the fluorescence of the NPY analogue [Trp³²]-NPY. Further, the position of NPY’s α-helix in the membrane was estimated from the intrinsic tyrosine fluorescence of NPY, from quenching experiments with spin-labelled phospholipids using [Trp³²]-NPY, and from ¹H magic-angle spinning NMR relaxation measurements using spin-labelled [Ala³¹, TOAC³²]-NPY. The results suggest that the immersion depth of NPY into the membrane is triggered by the membrane composition. The α-helix of NPY is located in the upper chain region of zwitterionic membranes but its position is shifted to the glycerol region in negatively charged membranes. For membranes composed of phosphatidylcholine and phosphatidylserine, an intermediate position of the α-helix is observed.