Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism, 1H NMR and molecular dynamics simulation

The conformation of a benzodiazepine-like decapeptide corresponding tothe YLGYLEQLLR fragment of a casein has been examined in a sodium dodecylsulfate micellar medium using circular dichroism, two-dimensional¹H NMR spectroscopy and restrained molecular dynamicssimulation. The decapeptide adopts an amphipathic 3₁₀-helicoidstructure in which theE⁶···R¹⁰ ionic bridgestabilizes the C-terminus.     

Relative free energy of binding between antimicrobial peptides and SDS or DPC micelles

We present relative binding free energy calculations for six antimicrobial peptide-micelle systems, three peptides interacting with two types of micelles. The peptides are the scorpion derived antimicrobial peptide (AMP), IsCT and two of its analogues. The micelles are dodecylphosphatidylcholine (DPC) and sodium dodecylsulphate (SDS) micelles. The interfacial electrostatic properties of DPC and SDS micelles are assumed to be similar to those of zwitterionic mammalian and anionic bacterial membrane interfaces, respectively. We test the hypothesis that the binding strength between peptides and the anionic micelle SDS can provide information on peptide antimicrobial activity, since it is widely accepted that AMPs function by binding to and disrupting the predominantly anionic lipid bilayer of the bacterial cytoplasmic membrane. We also test the hypothesis that the binding strength between peptides and the zwitterionic micelle DPC can provide information on peptide haemolytic activities, since it is accepted that they also bind to and disrupt the zwitterionic membrane of mammalian cells. Equilibrium structures of the peptides, micelles and peptide-micelle complexes are obtained from more than 300 ns of molecular dynamics simulations. A thermodynamic cycle is introduced to compute the binding free energy from electrostatic, non-electrostatic and entropic contributions. We find relative binding free energy strengths between peptides and SDS to correlate with the experimentally measured rankings for peptide antimicrobial activities, and relative free energy binding strengths between peptides and DPC to correlate with the observed rankings for peptide haemolytic toxicities. These findings point to the importance of peptide-membrane binding strength for antimicrobial activity and haemolytic activity.     

XII. Conformational studies of histidine-containing peptides in solution

The spectroscopic properties (uv, CD, nmr) of histidine, glycylhistidine, histidylglycine, glycylhistidylglycine have been investigated in water and methanol in the temperature range 200–320 K in order to obtain information about their conformational equilibria. This analysis has been carried out for the different ionic forms of the compounds, in order to evaluate the influence of the ionization state of the carboxyl, histidyl, and amino groups on the rotamer distribution of the histidyl side chain (as evaluated from proton nmr analysis) and on the overall molecule (as judged from CD spectra). On the basis of certain approximations and from the temperature dependence of the proton nmr resonance, the thermodynamic parameters (ΔH° and ΔS°) characterizing the conformational equilibrium of the hystidyl side chain have been evaluated for the different structures and ionization states. Relatively large entropy differences between the rotamers are obtained in some cases. The data of the sidechain rotamer population, as determined by nmr, have been analytically correlated with the CD data, and in the case of hystidine and histidylglycine in basic solution, first-approximation values for the ellipticity of the single conformers have been evaluated. Finally, in the example of glycylhistidine and histidylglycine in basic solution, it is shown how the data obtained from the different experimental approaches (nmr and CD), as well as from theoretical energy calculations, converge to characterize the most stable conformation in solution.     

Conformational analysis of bioactive peptides in reverse micelles as mimics of cell membrane environments

Summary Conformational preferences of secretin as a model peptide have been analyzed by CD and IR spectroscopy in reverse micelles of AOT/isooctane/water and compared to those in aqueous TFE, in SDS micelles and in DMPG vesicles. Among the systems examined, reverse micelles and phospholipid vesicles displayed almost identical conformational equilibria. Very high lipid-to-peptide ratios can be obtained in reverse micelles with full retention of optical transparency, even at millimolar peptide concentrations, thus indicating this system to be an interesting mimic of cell membrane environments for spectroscopic analysis of bioactive peptide conformations.Abbreviations TFE trifluoroethanol - SDS sodium dodecyl sulfate - DMPG dimyristoylphosphatidylglycerol - AOT bis(2-ethylhexyl)sulfosuccinate - CMC critical micellar concentration - VIP vasoactive intestinal peptide     

Conformational predictive studies on the activation segment of pancreatic procarboxypeptidases

Little is known about the conformation and evolutionary origin of the activation segment of pancreatic procarboxypeptidases. Analysis of the sequence and secondary structure propensities of these propeptide segments indicate that they contain two regions structurally related to the Ca2+-binding sites of the EF-hand protein family. This proposed homology could explain how (and why) carboxypeptidases developed such long (94 residues) activation peptides.     

1H-NMR of reverse micelles. II: Conformational studies of peptides and proteins in the AOT/water/isooctane system

The interaction of AOT reverse micelles with Met-enkephalin, the pancreatic secretory trypsin inhibitor (PSTI) and the epidermal growth factor (EGF) is examined by NMR methods and the three systems are compared. While Met-enkephalin adopts a folded conformation, PSTI appears to become highly flexible, suggestive of a non-specific interaction with the micelles. On the other hand, the EGF spectrum shows that, although the main globular features of the protein are retained in the presence of AOT, the C-terminal fragment has to rearrange its conformation when put in contact with the micelle wall.     

NMR studies of adrenocorticotropin hormone peptides in sodium dodecylsulfate and dodecylphosphocholine micelles: proline isomerism and interactions of the peptides with micelles

Three adrenocorticotropin hormone (ACTH) fragments (1-10, 1-24, and 11-24) have been studied in water and in sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles by nuclear magnetic resonance spectroscopy. The trans-cis isomerism at all three proline sites (at positions 12, 19, and 24) was found in the 11-24 segment of the peptide. The population of the cis isomers changes with the environment of the peptide. Specifically, the presence of the DPC micelle does not affect the trans-cis equilibrium in the 11-24 segment from that in water. In contrast, the presence of the SDS micelles decreases the population of the cis isomer at Pro(24), but increases its population at Pro(12) and Pro(19). The effect of SDS micelles on the trans-cis equilibrium at these proline sites was discussed. Intermolecular nuclear Overhauser effect (NOE) correlations between the ACTH peptides and the micelles were observed. These correlations occurred only in the 1-10 segment of the peptides, and the hydrophobic side chains contributed most to the intermolecular NOE. The intermolecular NOE pattern corroborates the suggestion that the 1-10 segment of the ACTH peptides bind to these micelles via a surface-binding mode, with most of the interactions coming from the insertion of the hydrophobic side chains.     

Conformational studies on the gramicidin A transmembrane channel in lipid micelles and liposomes

The interaction of gramicidin A with lysolecithin micelles and with lecithin liposomes is demonstrated by circular dichroism to result in several metastable conformational states. A stable state can be obtained after extensive heating when the gramicidin A was added dry or in ethanol solution to the phospholipid dispersion but the stable state is readily obtained when gramicidin A is added in a trifluoroethanol solution. The circular dichroism of the stable conformational state is characterized by negative ellipticity below 205 nm and principally by a positive 220 nm band on which is superposed a weak 230 nm band (the latter likely arising from tryptophan side chains). The stable conformational state is considered to be that of the functional transmembrane channel primarily on the basis of extensive studies on its interaction with sodium ions.     

Conformational studies on the gramicidin A transmembrane channel in lipid micelles and liposomes

The interaction of gramicidin A with lysolecithin micelles and with lecithin liposomes is demonstrated by circular dichroism to result in several metastable conformational states. A stable state can be obtained after extensive heating when the gramicidin A was added dry or in ethanol solution to the phospholipid dispersion but the stable state is readily obtained when gramicidin A is added in a trifluoroethanol solution. The circular dichroism of the stable conformational states is characterized by negative ellipticity below 205 nm and principally by a positive 220 nm band on which is superposed a weak 230 nm band (the latter likely arising from tryptophan side chains). The stable conformational state is considered to be that of the functional transmembrane channel primarily on the basis of extensive studies on its interaction with sodium ions.     

Conformational studies on peptides with proline in the right-handed alpha-helical region

The proline residues in proteins are known to play an important structural role. Recently, the role of a proline residue in the middle of right-handed alpha-helical segments of peptides has been the focus of attention. This role seems to be particularly important in the case of membrane proteins and in the tight packing of globular proteins. In the present study the right-handed alpha-helical region of the Ala-Pro dipeptide and of polypeptides containing this group have been investigated. Crystal structures of proline-containing alpha-helices from some proteins have been analyzed and energy minimization studies on some model fragments containing Ala-Pro in the right-handed alpha-helical conformation have been carried out using flexible geometry. The present calculations indicate that the right-handed alpha-helical region of conformational space is an energetically favored region that can also accommodate Ala-Pro in longer segments of right-handed alpha-helix. This is achieved due to minor variations in some of the internal parameters. Deviations in the backbone parameters of proline in the right-handed alpha-helix lead to a kink of about 23 degrees in the helix axis. These deviations have been characterized and a set of standard values has been suggested for producing such a kink. These values can be used for model building and as starting points for further minimization studies. Previous energy minimization studies have been done using rigid geometry. This may explain why the minimum for Ala-Pro in the right-handed alpha-helical region has not been recognized thus far.     

Conformational changes in NhaA Na+/H+ antiporter

Abstract

Na⁺/H⁺ antiporters play a primary role in Na⁺/H⁺ homeostasis in cells and many organelles and have long been drug targets. The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs (Trans membrane segments) are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site. Remarkably, inverted repeats containing interrupted helices with similar functional implications have since been observed in structures of other bacterial secondary transporters with almost no sequence homology. Finally, the structure reveals that NhaA is organized into two functional regions: a ‘pH sensor' – a cluster of amino acyl side chains that are involved in pH regulation; and a catalytic region that is 9 Å removed from the pH sensor. Alternative accessibility of the binding site to either side of the membrane, i.e., functional-dynamics, is the essence of secondary transport mechanism. Because NhaA is tightly pH regulated, structures of the pH-activated and ligand-activated NhaA conformations are needed to identify its functional-dynamics. However, as these are static snapshots of a dynamic protein, the dynamics of the protein both in vitro and in situ in the membrane are also required as reviewed here in detail. The results reveal two different conformational changes characterizing NhaA: One is pH-induced for NhaA activation; the other is ligand-induced for antiport activity.     

Structural Basis of H+-Dependent Conformational Change in a Bacterial MATE Transporter

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Segment TM7 from the cytoplasmic hemi-channel from VO-H+-V-ATPase includes a flexible region that has a potential role in proton translocation

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY (1)H-(1)H NMR spectra are obtained of sMTM7 dissolved in d(6)-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two alpha-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.     

Solution structures of the inactivation gate particle peptides of rat brain type-IIA and human heart sodium channels in SDS micelles.

The Ile-Phe-Met (IFM) motif located in the Ill-IV linker of voltage-gated sodium channels has been identified as a major component of the fast inactivation gate. If Gln was substituted for Phe, the role in the gate was disrupted completely. If Ile was replaced by Gln inactivation became slightly incomplete and if the Thr, which is adjacent to the IFM motif (-IFMT-), was replaced by Met, inactivation became much more incomplete than in the I/Q mutation, but not as vigorous as in the F/Q mutation. Previously, we studied the structures of the inactivation gate-related peptide (K1480-K1496 in rat brain type-IIA, MP-3A) and its F1489/Q substituted one (MP-4A) in SDS micelles and found that the conformational change of the IFM hydrophobic cluster due to the F/Q substitution may be a reason for disrupting the gate. In this study, in order to obtain supporting evidence for this view and also to further knowledge of the effect of I/Q and T/M mutations on the structure of the IFM cluster, we studied the structures of 11488Q [MP(rb)-3QFMT] and T1491M [MP(rb)-31FMM] substituted peptides. The fragment peptide K1477-K1493 [MP(hh)-3A] and its T1488M substituted peptide [MP(hh)-3IFMM] in the human heart sodium channel were also studied. It was found that the backbone structures around the IMF motif of MP-3A, MP(hh)-3A and MP(rb)-3QFMT resemble one another in such a manner that the residues Ile(Gln) and Thr are brought so close together that they form a unique type of lid to occlude the pore. In contrast, the residues between Ile and M1491 of MP(rb)-3IFMM or M1488 of MP(hh)-3IFMM were fairly far apart from each other. We conclude that Thr plays an important role in forming a structure of the IFM hydrophobic cluster for inactivation.     

Conformational requirement of signal sequences functioning in yeast: circular dichroism and 1H nuclear magnetic resonance studies of synthetic peptides

Recently, we have designed a series of simplified artificial signal sequences and have shown that a proline residue in the signal sequence plays an important role in the secretion of human lysozyme in yeast, presumably by altering the conformation of the signal sequence [Yamamoto, Y., Taniyama, Y., & Kikuchi, M. (1989) Biochemistry 28, 2728-2732]. To elucidate the conformational requirement of the signal sequence in more detail, functional and nonfunctional signal sequences connected to the N-terminal five residues of mature human lysozyme were chemically synthesized and their conformations in a lipophilic environment [aqueous trifluoroethanol (TFE) or sodium dodecyl sulfate micelles] analyzed by circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. The helix content of the peptides, including functional (L8, CL10) and nonfunctional (L8PL, L8PG, L8PL2) signal sequences, was estimated from CD spectra to be 40-50% and 60-70%, respectively, indicating that the helical structure is more abundant in the nonfunctional signal sequences. Two-dimensional NMR analyses in 50% TFE/H2O revealed that each peptide adopted a helical conformation throughout the sequence except for a few residues at the N- and C-termini. Furthermore, H-D exchange experiments indicated that the helical structure of the C-terminal region of the functional signal sequences (L8 and CL10) was less stable than that of the nonfunctional signal sequences (L8PL and L8PL2). On the basis of these results, a model was developed in which the functional signal sequence is inserted in the membrane with a helical conformation and the C-terminal helix unraveled in an extended conformational form through an interaction with the signal peptidase.     

Conformational studies on a peptide fragment representing the RNA-binding N-terminus of a viral coat protein using circular dichroism and NMR spectroscopy

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. The effects of ionic strength, addition of (oligo)phosphates and temperature on the conformation of this highly positively charged peptide containing six arginines and three lysines were studied. CD experiments show that the peptide has 15-18% alpha-helical conformation and about 80% random-coil conformation in the absence of inorganic salt at 25 degrees C, and 20-21% alpha-helical conformation under the same conditions at 10 degrees C. Addition of inorganic salts results in an increase of alpha-helix content, up to 42% in the presence of oligophosphate with an average chain length of 18 phosphates, which was used as an RNA analog. NMR experiments show that the alpha-helix formation starts in the region between Thr9 and Gln12, and is extended in the direction of the C terminus. Relaxation measurements show that binding to oligophosphates of increasing length results in reduced internal mobilities of the positively charged side chains of the arginyl and lysyl residues and of the side chain of Thr9 in the alpha-helical region. The alpha-helix formation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.     

Bioactive peptides: Conformational studies of [TYR4] cyclolinopeptide A

The solid state conformational analysis of [Tyr⁴] cyclolinopeptide A has been carried out by x-ray diffraction studies. The crystal structure of the monoclinic form, grown from a dioxane-water mixture [a = 9.849 (5) Å, b = 20.752 (4) Å, c = 16.728 (5) Å, β = 98.83 (3)°, space group P2₁, Z = 2], shows the presence of five intramolecular N-H? O?C hydrogen bonds, with formation of one C₁₇ ring structure, one α-turn (C₁₃), one inverse γ-turn (C₇), and two β-turns (C₁₀, one of type III and one of type 1). The Pro¹-Pro² peptide unit is cis (ω = 5°) all others are trans. The structure is almost superimposable with that of cyclolinopeptide A. The rms deviation for the atoms of the backbones is on the average 0.33 Å. © 1995 John Wiley & Sons, Inc.