Dicarboxylic acid analogs of Gramicidin A: Dimerization kinetics and single channel properties

Summary According to the model of Urry, the cation-permeable gramicidin channel is a dimeric helix formed by association of two peptide monomers linked at their amino ends. In this paper the channel properties of gramicidin analogs are described which have been obtained by chemical modification at the coupling site of the two half-channels. In these analogs the amino terminal-CHO group is replaced by-CO(CH₂) _n COOH(n=2, 3, 4, 5, 6). All analogs form conducting channels in black lipid membranes with the same general properties as found for gramicidin A. The observation that the channel-forming activity decreases with increasing pH is consistent with the notion that the half-channels are linked at the amino terminus. The channel lifetime of the different analogs varies between 2 msec and 50 sec, the longest lifetime being found for the compound withn=3. The single-channel conductance is always smaller than that of gramicidin A, but the reduction of depends on the nature of the permeable ion. Ion specificity was studied at 1m electrolyte by measuring the conductance for different permeable ions (Na⁺, K⁺, Cs⁺). The conductance ratio(Cs⁺)/(Na⁺) was found to vary between 2 and 10.5 for the different analogs.     

Formation of ionic channels in black lipid membranes by succinic derivatives of Gramicidin A

Summary Different succinyl derivatives of Gramicidin A were synthesized and their activity was investigated with different methods on lipid bilayer membranes. The succinyl derivatives of Gramicidin A can be classified as three different types, the O-succinyl derivative, the N-succinyl derivative and the N-O-succinyl derivative of Gramicidin A. An O-pyromellityl-N-succinyl gramicidin was synthesized which can be attributed to the latter class. It was found that O-succinyl gramicidin behaves like the unmodified Gramicidin A despite a charge effect on single-channel conductance, arising from the negative charge of the succinic residue, at the mouth of the channel. The activity of N-succinyl and N-O-succinyl gramicidin and of O-pyromellityl-N-succinyl-gramicidin depends strongly on the pH of the electrolyte solution. It is demonstrated that at low pH (5) the N-succinyl derivatives show high activity, whereas at high pH (7) the activity is sharply reduced or disappears totally. From these experiments it can be concluded that, for the formation of a dimeric gramicidin channel, the hydrogen of the formyl group can be replaced by a protonated carboxylic group of a succinic residue.Further results, obtained by measurement of the single-channel conductance and of the reaction rate constants for the channel formation, are discussed in terms of the structural basis of the single stranded model for the gramicidin channel. On this basis the double stranded helix can be, excluded and an interesting head-to-head single stranded (_L,D) helical channel is described which contains carboxyl groups at the head-to-head junction.     

The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3m-cort binding of HPK1 proline-rich peptide

SH3 domains are probably the most abundant molecular-recognition modules of the proteome. A common feature of these domains is their interaction with ligand proteins containing Pro-rich sequences. Crystal and NMR structures of SH3 domains complexes with Pro-rich peptides show that the peptide ligands are bound over a range of up to seven residues in a PPII helix conformation. Short proline-rich peptides usually adopt little or no ordered secondary structure before binding interactions, and consequently their association with the SH3 domain is characterized by unfavorable binding entropy due to a loss of rotational freedom on forming the PPII helix. With the aim to stabilize the PPII helix conformation into the proline-rich decapeptide PPPLPPKPKF (P2), we replaced some proline residues either with the 4(R)-4-fluoro-l-proline (FPro) or the 4(R)-4-hydroxy-l-proline (Hyp). The interactions of P2 analogues with the SH3 domain of cortactin (SH3_m-cort) were analyzed by circular dichroism spectroscopy, while CD thermal transition experiments have been used to determine their propensity to adopt a PPII helix conformation. Results show that the introduction of three residues of Hyp efficiently stabilizes the PPII helix conformation, while it does not improve the affinity towards the SH3 domain, suggesting that additional forces, e.g., electrostatic interactions, are involved in the SH3_m-cort substrate recognition.     

Raman and infrared spectroscopic study of gramicidin A conformations

Raman scattering and infrared spectroscopic techniques were used to study the vibrational spectrum and conformation of the membrane channel protein gramicidin A in the solid state, in organic solutions and, using Raman scattering only, in a phospholipid environment. The investigation also includes measurements on head- and tail-group-modifled gramicidin A and a potassium thiocyanate-gramicidin A complex. Tentative identification of the molecular vibrations is proposed on the basis of the data on model compounds. The existence of four distinct conformations of the gramicidin A chain is established: conformation I present in the solid state, and CH₃OH and CD₃OD solutions; conformation II present in films cast from CHCl₃ solution; conformation III present in (CH₃)₂SO and (CD₃)₂SO solutions at concentrations below 0.5 m gramicidin A; and conformation IV present in the potassium thiocyanate-gramicidin A complex. The data obtainable on a gramicidin A-phospholipid suspension indicate a gramicidin A conformation in this environment corresponding either to the conformation I or II. The details of the spectra in the amide I region are shown to be consistent with a β-parallel hydrogen-bonded π_LD helix for conformational I, in terms of the polypeptide vibrational calculations of Nevskaya and co-workers. Conformation II is found to be consistent with an antiparallel double-stranded π_LD helix, while conformations III and IV probably have π-helical structures with larger channel diameters. The data on head- and tail-modified gramicidin A molecules indicate that their conformations are only slightly different from that of gramicidin A in conformation I.     

Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), ¹³C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of ¹³C=O isotope-labeled (Ala-Gly-Gly)₁₀. The torsion angles were determined to be (, ) = (–90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 3₁ helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3₁ helix of (Ala-Gly-Gly)₁₀ does not change by formic acid treatment although (Ala-Gly)₁₅ easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3₁ helix conformation of (Ala-Gly-Gly)₁₀ is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)₁₀ were also determined as (, ) = (–90°, 150° ) and this peptide is also considered to take 3₁ helix conformation.     

1/f noise in black lipid membranes induced by ionic channels formed by chemically dimerized gramicidin A

Summary The noise behavior of lipid bilayer membranes, doped with a chemically dimerized gramicidin A, was investigated. In contrast to normal gramicidin A, which generates a Lorentzian type power spectrum due to the formation and disappearance of conducting dimers, the current power spectrum densityS _m (f) obtained with this gramicidin A derivative showed over several orders of magnitude a clear 1/f behavior. The intensity of this 1/f component was analyzed as a function of the membrane-applied voltage, membrane resistance, electrolyte concentration, and composition. The relationship between the meansquare fluctuation in current and the membrane current mean value was found to follow Hooge's equation, i.e., I ²=I _m ² /N f whereN is the number of channels and is a constant equal to 1.0×10^–2. It is suggested that a 1/f type noise was observed because the chemically dimerized form of gramicidin A produces long lasting cation selective channels.     

Electrical properties of the rabbit urinary bladder assessed using gramicidin D

Summary Recently, antibiotics have enjoyed widespread usage as tools in studies of epithelial transport. In the present study we assess the usefulness of the pore-forming antibiotic gramicidin D as a means for probing the electrical properties of the tight epithelium rabbit urinary bladder. Addition of 50 M gramicidin to the mucosal bath (either a NaCl or KCl Ringer's solution) led to a large irreversible increase in the transepithelial conductance (G _T ) within 800 sec.G _T increased by approximately 1200% and 500% in KCl and NaCl Ringer's solutions, respectively. Microelectrode measurements of the resistance ration (the ration of apical membrane resitance to basolateral membrane resistance) showed that apical membrane resistance is dereased by the drug. Measurements of the basolateral membrane resistance (R _bl ) and tight junctional resistance (R _j ) using a new and independent method (based on the perturbation of basolateral membrane electrogenic Na⁺ pump) demonstrated thatR _bl andR _j were unaffected, suggesting that the effects of gramicidin are restricted to the apical membrane for periods of at least 2 hours after drug addition. The selectivity of the gramicidin-induced permeability in the apical membrane was calculated from measurements of the apical membrane potential after ion substitutions using a modified version of the constant field equation. The selectivity sequence for cations was Cs⁺>K⁺>Na⁺>Li⁺>choline. Unlike the commonly used polyene antibiotics nystatin and amphotericin B, gramicidin did not induce a significant Cl^– permeability. In addition, the dose-response curve had a slope of 1. A method is described for calculating membrane resistances directly from transepithelial measurements under some conditions of gramicidin use, without requiring the use of microlectrode measurements.     

The recognition of mismatched base pairs in DNA by DNase I from Ustilago maydis.

Summary The activity of Ustilago maydis DNAse I, an enzyme implicated in genetic recombination, on DNA substrates containing unpaired or mismatched bases, was examined. The enzyme nicked supercoiled PM-2 molecules, converting these to relaxed circular and linear molecules. Discrete double stranded linear fragments smaller than unit length were also observed after digestion at high enzyme concentration. Heteroduplex molecules were constructed using 80 bacteriophage derivatives which contained single base substitutions within the E. coli tRNA ₁ ^tyr gene. Single and double stranded nicking at or near the single mismatched site was observed with three out of the five pairs of heteroduplexes.     

Influence of a Transmembrane Protein on the Permeability of Small Molecules Across Lipid Membranes

The influence of the nonchannel conformation of the transmembrane protein gramicidin A on the permeability coefficients of neutral and ionized α-X-p-methyl-hippuric acid analogues (XMHA) (X = H, OCH₃, CN, OH, COOH, and CONH₂) across egg-lecithin membranes has been investigated in vesicle efflux experiments. Although 10 mol% gramicidin A increases lipid chain ordering, it enhances the transport of neutral XMHA analogues up to 8-fold, with more hydrophilic permeants exhibiting the greatest increase. Substituent contributions to the free energies of transfer of both neutral and anionic XMHA analogues from water into the bilayer barrier domain were calculated. Linear free-energy relationships were established between these values and those for solute partitioning from water into decadiene, chlorobutane, butyl ether, and octanol to assess barrier hydrophobicity. The barrier domain is similar for both neutral and ionized permeants and substantially more hydrophobic than octanol, thus establishing its location as being beyond the hydrated headgroup region and eliminating transient water pores as the transport pathway for these permeants, as the hydrated interface or water pores would be expected to be more hydrophilic than octanol. The addition of 10 mol% gramicidin A alters the barrier domain from a decadiene-like solvent to one possessing a greater hydrogen-bond accepting capacity. The permeability coefficients for ionized XMHAs increase with Na⁺ or K⁺ concentration, exhibiting saturability at high ion concentrations. This behavior can be quantitatively rationalized by Gouy-Chapman theory, though ion-pairing cannot be conclusively ruled out. The finding that transmembrane proteins alter barrier selectivity, favoring polar permeant transport, constitutes an important step toward understanding permeability in biomembranes. Received: 12 July 1999/Revised: 20 October 1999     

Conformational Polymorphism and Extensibility of DNA Quadruplexes Formed by d(GT)nRepeats

We showed earlier that oligonucleotides 3"-d(GT)₅-pO(CH₂CH₂O)₃p-d(GT)₅-3" form bimolecular quadruplexes with parallel orientation of their strands, which are held by guanine quartets alternating with unpaired thymines (GT quadruplex). This work deals with the conformational polymorphism and extensibility of G quadruplexes in complex with molecules of an intercalating agent ethidium bromide (EtBr). A cooperative mechanism of EtBr binding to the GT quadruplex was revealed. The binding constant K= (3.3 ± 0.1)·10⁴M^–1, cooperativity coefficient = 2.5 ± 0.2, and maximal amount of EtBr molecules intercalated in GT quadruplex (N= 8) were determined. It was proved experimentally by analysis of adsorption isotherms and theoretically by mathematical modeling that the GT quadruplex is capable of double extension, which is indicative of the high elasticity of this four-stranded helix. Two most stable conformations of GT quadruplexes with thymine residues intercalated and/or turned outside were found by mechanico-mathematical modeling. The equilibrium is shifted toward the conformation with the looped out thymine residues upon intercalation of EtBr molecules into the GT quadruplex.     

Molecular definition of an elusive third HLA-A9 molecule: HLA-A9.3

The HLA-A9 family has been characterized as possessing two well defined specificities; HLA-A23 and A24. Serological studies have suggested the presence of a third member of this family HLA-A9.3, however there is doubt surrounding the existence of this specificity. HLA-A23, A24, and the putative A9.3 proteins were analyzed biochemically by immunoprecipitation and isoelectric focusing. Both HLA-A24 and A9.3 have identical isoelectric points whereas A23 is different. We have sequenced cDNA encoding HLA-A23, A24, and A9.3. From the observed protein sequences, we found A9.3 to differ from A24 by two amino acid substitutions located in the ₂ helix of the class I molecule. These substitutions are expected to significantly change the shape of the peptide binding cleft.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M64740 (HLA-A ^*2402); M64741 (HLA-A ^*2403); M64742 (HLA-A ^*2301). Address correspondence and offprint requests to: P. Parham.     

Spectroscopic studies on the structure of poly(8-bromoadenylic acid): Effect of glycosidic torsion angle on the conformation and flexibility in polyribonucleotides

The helix–coil transition and conformational structure of poly(8-bromoadenylic acid) [poly(8BrA)] have been investigated using ¹H- and ¹³C-nmr, CD, and ir spectroscopy. The results have been compared with the structure of the related 5′-mono- and polynucleotides. The chemical shifts of H(2′), H(3′), C(2′), and C(3′) nmr signals show an interesting correlation with both the puckering of ribose ring and glycosidic bond torsion angle. Poly(8BrA) shows an upfield shift of the C(3′) signal and a downfield shift of the H(3′) signal compared to the chemical shifts in poly(A). These shifts are consistent with a C(3′) endo-syn conformation for poly(8BrA). A similar effect has been reported previously and is also observed here on the C(2′) and H(2′) signals when the preferred conformation is C(2′)endo-syn (e.g., in 5′-8BrAMP). The chemical-shift parameters thus act as a probe for studying syn ? anti and N ? S equilibria in solutions. The three-bond ¹H-′¹³C coupling constants between H(1′) and C(8) and C(4) have been measured in poly(8BrA) and 5′-8BrAMP and their structural implications have been discussed. The observed preference of a C(3′)endo-syn conformation for poly(8BrA), coupled with other evidence, throws doubt on the validity of a correlation previously reported whereby a syn conformation is associated with a C(2′)endo ribose pucker. The backbone conformation of randomly coiled poly(8BrA) is very similar to the structures found in polyribonucleotides: poly(A) and poly(U). All three polymers show strong preferences for the backbone angles found in RNA helices. The CD spectrum of poly(8BrA) has a striking relationship to that of poly(A). The signs of all extrema are inverted, and the magnitudes are related by a constant factor. We suggest that these differences result from a change in the angle between coupled transition moment vectors in the two polymers. Infrared spectra of poly(8BrA) in H₂O and D₂O solution are reported for the frequency range below 1400 cm^?1. The antisymmetric >PO stretching vibration is observed at an unusually low frequency in the helix (1214 cm^?1). The symmetric >PO stretch occurs at ～1095 cm^?1 but is not resolved from a ring vibration near this frequency. A conformationally sensitive band, characteristic of helical RNA structures, is observed at 817 cm^?1 and disappears when the helix is melted. This observation confirms the conclusion that ordered poly(8BrA) has a regular helical structure with an RNA backbone conformation. A stereochemical explanation is provided for the failure of poly(8BrA) (or other syn polymers) to form double helices with anti-polyribonucleotides.     

Two-dimensional NMR study of the conformation of (34–65)bacterioopsin polypeptide in SDS micelles

Summary The conformation of the synthetic 32-residue polypeptide, an analog of the membrane spanning segment B (residues 34-65) ofHalobacterium halobium bacteriobpsin, incorporated into perdeuterated sodium dodecyl sulfate micelles in the presence of trifluoroethanol was investigated by¹H NMR spectroscopy. The spectrum resonances were assigned by means of phase-sensitive DQF-COSY, TOCSY and NOESY techniques. Interproton nuclear Overhauser effects and deuterium exchange rates of individual NH groups were derived from two-dimensional NMR spectra. Analysis of the obtained data showed that segment B has a right-handed a-helical stretch from Lys⁴¹ to Leu⁶² with a kink at Pros⁵⁰. The-helix in the C-terminal part is terminated at Gly⁶³, which adopts a conformation typical of amino acid residues in a left-handed helix. The N-terminal part (residues 34–40) has no ordered conformation. NMR data are provided for comparison of the segment B conformation in the isotropic system of an organic solvent, in SDS micelles and in the purple membrane bacterioopsin. Factors affecting the conformation of membrane spanning segment B in various milieus are discussed.Dedicated to the memory of Professor V.F. Bystrov     

Large scale conformational transitions in β‐structural motif of gramicidin A: kinetic analysis based on CD and FT‐IR data

Gramicidin A (gA) is a polypeptide antibiotic, which forms dimeric channels specific for monovalent cations in artificial and biological membranes. It is a polymorphic molecule that adopts a unique variety of helical conformations, including antiparallel double‐stranded ↑↓β5.6 or ↑↓β7.2 helices (number of residues per turn) and a single‐stranded β6.3 helix (the ‘channel form’). The behavior of gA‐Cs⁺ complex in the micelles of TX‐100 was studied in this work. Transfer of the complex into the micelles activates a cascade of sequential conformational transitions monitored by CD and FT‐IR spectroscopy: At the first step after Cs⁺ removal, the RH ↑↓β5.6 helix is formed, which has been discussed so far only hypothetically. Kinetics of the transitions was measured, and the activation parameters were determined. The activation energies of the ↑↓β5.6 → β‐helical monomer transition in dioxane and dioxane/water solutions were also measured for comparison. The presence of water raises the transition rate constant ~10³ times but does not lead to crucial fall of the activation energy. All activation energies were found in the 20–25 kcal/mol range, i.e. much lower than would be expected for unwinding of the double helix (when 28 H‐bonds are broken simultaneously). These results can be accounted for in the light of local unfolding (or ‘cracking’) model for large scale conformational transitions developed by the P. G.Wolynes team [Miyashita O, Onuchic JN, Wolynes PG. Proc. Natl. Acad. Sci. USA 2003; 100: 12570‐12575.]. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and preliminary conformational analysis of TOAC spin‐labeled analogues of the medium‐length peptaibiotic tylopeptin B

A set of analogues of the 14‐residue peptaibol tylopeptin B, containing the stable free‐radical 4‐amino‐1‐oxyl‐2,2,6,6,‐tetramethylpiperidine‐4‐carboxylic acid (TOAC) at one or two selected positions, was synthesized by the solid‐phase methodology. A solution conformational analysis performed by FTIR absorption and CD suggests that, in membrane‐mimicking solvents, the labeled tylopeptin B analogues preserve the helical propensity of the parent peptide, with a preference for the α‐helix or the 3₁₀‐helix type depending upon the nature of the solvent. In aqueous environment, the spin‐labeled analogues present a higher content of helical conformation as a consequence of the strong helix promoter effect of the conformationally constrained TOAC residue. We observed a progressive increase of the quenching effect of the nitroxyl radical on the fluorescence of the N‐terminal tryptophan as TOAC replaces the Aib residue at positions 13, 8, and 4, respectively. A membrane permeabilization assay performed on two selected analogues, TOAC⁸‐ and TOAC¹³‐tylopeptin B, showed that the labeled peptides exhibit membrane‐modifying properties comparable with those of the natural peptaibiotic. We conclude that our TOAC paramagnetic analogues of tylopeptin B are good models for a detailed ESR investigation of the mechanism of membrane permeabilization induced by medium‐length peptaibiotics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Design of analogues of parathyroid hormone: A conformational approach

An approach to the design of peptide-hormone analogues in which amino acid substitutions are based on predicted effects on secondary structure was investigated. The structural requirements for parathyroid-hormone (PTH) action are distinct from the determinants necessary for receptor binding alone without subsequent activation of adenylate cyclase. Two analogues of PTH containing substitutions in the principal binding domain of PTH, the region 25–34, were synthesized by the solid-phase method and evaluated for bioactivity. The sequence 25–34 was predicted to have nearly equal conformational potential for both -helix and -sheet using Chou and Fasman parameters. A previously studied analogue, [Tyr³⁴]bPTH(1–34) amide, containing substitutions in this region, was more active than was bPTH-(1–34). The substitution of tyrosine for phenylalanine at position 34 in this analogue is predicted to promote -sheet conformation. The analogues [Ile²⁸, Tyr³⁰, Tyr³⁴]bPTH-(1–34) amide and [Arg³², Tyr³⁴]bPTH-(1–34) amide each contain substitutions predicted to further enhance or stabilize -sheet formation. The solution conformation of these analogues, determined by circular dichroism studies in an aqueous buffer and an organic solvent, indicated promotion of -sheet secondary structural content in both analogues in a hydrophobic environment chosen to simulate that of the interaction of the peptide and the membrane receptor. In contrast, the native sequence lacks -structure. Biological activity of these analogues in the rat renal adenylate cyclase assay in vitro and binding affinity in a radioreceptor assay were threefold those of unsubstituted PTH-(1–34). Peptide analogue design based on conformational prediction, rather than substitution of primary structure alone, offers an attractive alternative approach to the development of hormone analogues and antagonists.     

Solution conformation of a tetradecapeptide stabilized by two di‐n‐propyl glycine residues

The solution conformation of a designed tetradecapeptide Boc‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐OMe (Dpg‐14) containing two di‐n‐propyl glycine (Dpg) residues has been investigated by ¹H NMR and circular dichroism in organic solvents. The peptide aggregates formed at a concentration of 3 mM in the apolar solvent CDCl₃ were broken by the addition of 12% v/v of the more polar solvent DMSO‐d₆. Successive N_iH N_i+1H NOEs observed over the entire length of the sequence in this solvent mixture together with the observation of several characteristic medium‐range NOEs support a major population of continuous helical conformations for Dpg‐14. Majority of the observed coupling constants ( ) also support ? values in the helical conformation. Circular dichroism spectra recorded in methanol and propan‐2‐ol give further support in favor of helical conformation for Dpg‐14 and the stability of the helix at higher temperature. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Order–Disorder conformation change of xanthan in 0.01M aqueous sodium chloride: Dimensional behavior

Weight-average molecular weights M_w, second virial coefficients, and z-average radii of gyration 〈S²〉 were determined by light scattering as a function of temperature T for four sodium salt samples of xanthan in 0.01M aqueous NaCl, in which the polysaccharide undergoes an order–disorder conformation change with increasing T. The data for 〈S²〉 and M_w at 25 and 80°C, the lowest and highest temperatures studied, confirmed the previous conclusion that the predominant conformation at the former T, i.e., in the ordered state, is a double helix, while that at the latter T, i.e., in the disordered state, is a dimerized coil expanded by electrostatic repulsions between charged groups of the polymer. As T was increased from 25 to 80°C, 〈S²〉 sigmoidally decreased or increased depending on the dimer's molecular weight. This temperature dependence of 〈S²〉 and that determined elsewhere for a high molecular weight sample were found to be described almost quantitatively by a simple dimer model in which the double helix melts from both ends, when the double-helical fraction in the dimer at a given T estimated previously from optical rotation data was used.     

Spatial structure of (34–65)bacterioopsin polypeptide in SDS micelles determined from nuclear magnetic resonance data

Summary The spatial structure of a synthetic 32-residue polypeptide, an analog of the membrane-spanning segment B (residues 34–65) of bacterioopsin ofHalobacterium halobium, incorporated into perdeuterated sodium dodecyl sulfate micelles, was determined from¹H NMR data. The structure determination included the following steps: (1) local sructure analysis; (2) structure calculations using the distance geometry program DIANA; (3) systematic search for energetically allowed side-chain rotamers consistent with NOESY crosspeak volumes; (4) random generation of peptide conformations in allowed conformational space. The obtained structure has a righ-handed -helicl region from Lys⁴¹ to Leu⁶² with a kink of 27 at Pro⁵⁰. The C-cap Gly⁶³ adopts a conformation with =87±6, =43±10^o typical to a left-handed helix. The N-terminal part (residues 34–40) is exposed to the aqueous phase and lacks an ordered conformation. The secondary structure of segment B in micelles is consistent with the high-resolution electron cryomicroscopy model of bacteriorhodopsin (Henderson et al. (1990)J. Mol. Biol.,213, 899–929).     

Characterization of Aβ aggregation mechanism probed by congo red

β-Amyloid peptide ^{1 1}These authors contributed equally to this work. Communicated by Ramaswamy H. Sarma (Aβ) aggregates are toxic to neuron and the main cause of Alzheimer’s disease (AD). The role of congo red (CR) on Aβ aggregation is controversial in aqueous solution. Both prevention and promotion of Aβ aggregation have been proposed, suggesting that CR may interact with Aβ of different structural conformations resulting in different effects on Aβ aggregation behavior. CR with these characteristics can be applied to probe the molecular mechanism of Aβ aggregation. Therefore, in the present study, we used CR as a probe to study the Aβ aggregation behavior in sodium dodecyl sulfate (SDS) condition. Our results show that Aβ₄₀ adopts two short helices at Q15-S26 and K28-L34 in the SDS environment. CR can interact with the helical form of Aβ₄₀, and the main interaction site is located at the first helical and hydrophobic core region, residues 17–25, which is assigned as a discordant helix region. Furthermore, CR may prevent Aβ₄₀ undergoing α-helix to β-strand conversion, and therefore aggregation through stabilizing the helical conformation of discordant helix in SDS environment, suggesting that the discordant helix plays a key role on the conformational stabilization of Aβ. Our present study implies that any factors or molecules that can stabilize the discordant helical conformation may also prevent the Aβ aggregation in membrane associated state. This leads to a new therapeutic strategy for the development of lead compounds to AD.