Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocyclopropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS （LeACS2） into 52-, 50- and 49-kDa truncated isoforms in ripening tomato （Lycopersicon esculentum Mill. cv. Cooperation 903） fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted （26-50 amino acids） LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain （H14） and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.     

Structure–activity relationship of a novel pentapeptide with cancer cell growth‐inhibitory activity

We previously reported that yamamarin, a pentapeptide with an amidated C‐terminus (DILRG‐NH₂) isolated from larvae of the silkmoth, and its palmitoylated analog (C16‐DILRG‐NH₂) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure–activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C‐terminal part (‐RG‐NH₂) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG‐NH₂ and C16‐DILRG‐NH₂ revealed that the N‐terminal palmitoyl group of C16‐DILRG‐NH₂ did not affect the conformation of the C‐terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Assembly of binding loops on aromatic templates as VCAM‐1 mimetics

The design and synthesis of cyclic mimetics of VCAM‐1 protein that reproduce the integrin‐binding domain are presented. The unprotected peptide precursor 37 – 43 , Thr‐Gln‐Ile‐Asp‐Ser‐Pro‐Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C‐ and N‐terminal oximes resulting in a mixture of four isomeric forms due to syn–anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene‐derived template was found to inhibit the VCAM‐1/VLA‐4 interaction more efficiently than previously reported for sulfur‐bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37 – 43 in the design of synthetic inhibitors of the VCAM‐1/integrin interaction. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

δ-Selective Opioid Peptides Containing a Single Aromatic Residue in the Message Domain: An NMR Conformational Analysis

The sequence of deltorphin I, a δ-selective opioid agonist, has been systematically modified by inserting conformationally constrained C^α,α disubstituted apolar residues in the third position. As expected, substitution of Phe with Ac₆c, Ac₅c and Ac₃c yields analogues with decreasing but sizeable affinity. Surprisingly, substitution with Aib yields an analogue with almost the same binding affinity of the parent compound but with a greatly increased selectivity. This is the first case of a potent and very selective opioid peptide containing a single aromatic residue in the message domain, that is, only Tyr¹. Here we report a detailed conformational analysis of [Aib³]deltorphin I and [Ac₆c³]deltorphin I in DMSO at room temperature and in a DMSO/water cryomixture at low temperature, based on NMR spectroscopy and energy calculations. The peptides are highly structured in both solvents, as indicated by the exceptional finding of a nearly zero temperature coefficient of Val⁵ NH resonance. NMR data cannot be explained on the basis of a single structure but it was possible to interpret all NMR data on the basis of a few structural families. The conformational averaging was analysed by means of an original computer program that yields qualitative and quantitative composition of the mixture. Comparison of the preferred solution conformations with two rigid δ-selective agonists shows that the shapes of [Aib³]deltorphin I and [Ac₆c³]deltorphin I are consistent with those of rigid agonists and that the message domain of opioid peptides can be defined only in conformational terms.     

Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational analyses of cyclic hexapeptide analogs of somatostatin containing arylalkyl peptoid and naphthylalanine residues

We report the conformational analysis by ¹H‐NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of peptoid analogs of the cyclic hexapeptide c‐[Phe¹¹‐Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] L‐363,301 (the numbering refers to the positions in native somatostatin). The compounds c‐[Phe¹¹‐Nphe⁶‐Nal⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nphe ⁶ ‐ Nal ⁷ analog 1 ), c‐[Nal¹¹‐Nphe⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nal ¹¹ ‐ Nphe ⁶ analog 2 ) and c‐[Phe¹¹‐Nnal⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nnal ⁶ analog 3 ), where Nphe denotes N‐benzylglycine and Nnal denotes N‐(1‐naphthylmethyl)glycine, are subjected to SAR studies in order to investigate the influence of the bulky naphthyl aromatic ring on the conformation. The Nal ¹¹ ‐ Nphe ⁶ and Nphe ⁶ ‐Nal ⁷ analogs exhibit potent binding to the hsst2, hsst3 and hsst5 receptors, whereas the Nnal ⁶ analog has decreased binding affinity to all receptors but is more selective towards the hsst2 than the other two analogs and L‐363,301. The conformational search employing distance geometry, energy minimization and molecular dynamic simulations gives insight into the conformational flexibility of these analogs. The molecules adopt both cis and trans orientations of the peptide bond between residues 11 and 6. The cis isomers of these analogs adopt type II′ β‐turns with d ‐Trp in the i+1 position and type VIa β‐turns with the cis peptide bond between residues 6 and 11. The results of free and distance restrained molecular dynamics simulations at 300 K indicate that the Nphe ⁶ ‐Nal ⁷ and Nal ¹¹ ‐Nphe ⁶ compounds adopt a preferred backbone conformation which can be described as ‘folded’ about residues 7 and 10. The Nnal ⁶ analog, which binds less effectively to the hsst receptors, has a more flexible backbone structure than the Nal ¹¹ ‐Nphe ⁶ and Nphe ⁶ ‐Nal ⁷ analogs and prefers a ‘flat’ structure with regard to the orientations about Phe⁷ and Thr¹⁰ during molecular dynamics simulations. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

The wild‐type HIV‐1 capsid protein (CA) self‐assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV‐1. Mass‐per‐length values of CA assemblies determined by dark‐field transmission electron microscopy indicate a variety of structures, ranging from single‐wall tubes to multiwall tubes that approximate solid rods. Two‐dimensional (2D) solid state ¹³C? ¹³C and ¹⁵N? ¹³C NMR spectra of uniformly ¹⁵N,¹³C‐labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3‐¹³C₂‐glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N‐terminal (NTD) and C‐terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self‐assembly of full‐length CA into tubes. 2D ¹H‐¹³C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the α‐helices of NTD and CTD and from the N‐ and C‐terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Enantioselective recognition of carboxylic acids by novel fluorescent triazine‐based thiazoles

Hydrogen bonding and π‐π interactions take special part in the enantioselectivity task. In this regard, because of having both hydrogen acceptor and hydrogen donor groups, melamine derivatives become more of an issue for enantioselectivity. In the light of such information, triazine‐based chiral, fluorescence active novel thiazole derivatives L1 and L2 were designed and synthesized from (S)‐(?)‐2‐amino‐1‐butanol and (1S,2R)‐(+)‐2‐amino‐1,2‐diphenylethanol. The structural establishment of these compounds was made by spectroscopic methods such as FTIR, ¹H, and ¹³C NMR. While the solution of these compounds in DMSO did not show any fluorescence emission, it was observed that the emission increased 44‐fold for L1 and 55‐fold for L2 in 95% water, similar to the aggregation‐induced emission (AIE) characterized compounds. In this regard, enantioselective capabilities of these compounds against carboxylic acids were tested, and in experiments carried out at a ratio of 40/60 DMSO/H₂O, it was determined that R‐2ClMA increased the fluorescence emission of L1 chiral receptor by 2.59 times compared to S‐isomer.     

Isostructural unbranched alkyl‐chains as tools for stabilizing β‐turn structure

To investigate the structural role played by isostructural unbranched alkyl‐chains on the conformational ensemble and stability of β‐turn structures, the conformational properties of a designed model peptide: Plm‐Pro‐Gly‐Pda ( 1 , Plm: H₃C—(CH₂)₁₄—CONH—; Pda: —CONH— (CH₂)₁₄—CH₃) have been examined and compared with the parent peptide: Boc‐Pro‐Gly‐NHMe ( 2 , Boc: tert‐butoxycarbonyl; NHMe: N‐methylamide). The characteristic ¹³C NMR chemical‐shifts of the Pro C^β and C^γ resonances ascertained the incidence of an all‐trans peptide‐bond in low polarity deuterochloroform solution. Using FTIR and ¹H NMR spectroscopy, we establish that apolar alkyl‐chains flanking a β‐turn promoting Pro‐Gly sequence impart definite incremental stability to the well‐defined hydrogen‐bonded structure. The assessment of ¹H NMR derived thermodynamic parameters of the hydrogen‐bonded amide‐NHs via variable temperature indicate that much weaker hydrophobic interactions do contribute to the stability of folded reverse turn structures. The far‐UV CD spectral patterns of 1 and 2 in 2,2,2‐trifluoroethanol are consistent with Pro‐Gly specific type II β‐turn structure, concomitantly substantiate that the flanking alkyl‐chains induce substantial bias in enhanced β‐turn populations. In view of structural as well as functional importance of the Pro‐Gly mediated secondary structures, besides biochemical and biological significance of proteins lipidation via myristoylation or palmytoilation, we highlight potential convenience of the unbranched Plm and Pda moieities not only as main‐chain N‐ and C‐terminal protecting groups but also to mimic and stabilize specific isolated secondary and supersecondary structural components frequently observed in proteins and polypeptides. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 419–426, 2013.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Further studies on the antiviral activity of alloferon and its analogues

The subject of our studies was the synthesis, biological evaluation, and conformational studies of insect tridecapeptide alloferon (H‐His‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH) and its analogues such as: [des‐His¹]‐, [Lys¹]‐, [Arg¹]‐, and [Ala¹]‐alloferon. These peptides were synthesized to check the influence of the His residue at position 1 of the alloferon chain on its antiviral activity. Two aspects of the biological effects of these peptides were determined: (i) the cytotoxicity in vitro in the Vero, LLC‐MK2, and HEp‐2 cell lines, and (ii) the antiviral activity in vitro in respect to DNA and RNA viruses. We found that alloferon inhibited the herpes virus multiplication and failed to affect the coxsackie virus replication, whereas [Lys¹]‐alloferon exhibited a high inhibitory action towards both viruses. Moreover, the peptides did not show any cytotoxic activity against the Vero, LLC‐MK2, and HEp‐2 cells. The preliminary circular dichroism conformational studies showed that the peptides investigated seem to prefer an unordered conformation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Conformation dependence of the CαDα stretch mode in peptides. II. Explicitly hydrated alanine peptide structures

Our previous studies of the potential utility of the C^αD^α stretch frequency, ν(CD), as a tool for determining conformation in peptide systems (Mirkin and Krimm, J Phys Chem A 2004, 108, 10923–10924; 2007, 111, 5300–5303) dealt with the spectroscopic characteristics of isolated alanine peptides with α_R, β, and polyproline II structures. We have now extended these ab initio calculations to include various explicit‐water environments interacting with such conformers. We find that the structure‐discriminating feature of this technique is in fact enhanced as a result of the conformation‐specific interactions of the bonding waters, in part due to our finding (Mirkin and Krimm, J Phys Chem B 2008, 112, 15268) that C^α? D^α…O(water) hydrogen bonds can be present in addition to those expected between water and the CO and NH of the peptide groups. In fact, ν(CD) is hardly affected by the latter bonding but can be shifted by up to 70 cm^?1 by the former hydrogen bonds. We also discuss the factors that will have to be considered in developing the molecular dynamics (MD) treatment needed to satisfactorily take account of the influence of outer water layers on the structure of the first‐layer water molecules that hydrogen bond to the peptide backbone. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 791–800, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

MAP dendrimer elicits antibodies for detecting rat and mouse GH‐binding proteins

The membrane‐bound rat GH‐R and an alternatively spliced isoform, the soluble rat GH‐BP, are comprised of identical N‐terminal GH‐binding domains; however, their C‐terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent MAP dendrimer with four identical branches of a C‐terminal peptide sequence of the rat GH‐BP (GH‐BP_263–279) was synthesized and used as an immunogen in rabbits. Solid‐phase peptide synthesis of four GH‐BP_263–279 segments onto a tetravalent Lys₂‐Lys‐β‐Ala‐OH core peptide was carried out using Fmoc chemistry. The mass of the RP‐HPLC‐purified synthetic product, 8398 Da, determined by ESI‐MS, was identical to expected mass. Three anti‐rat GH‐BP_263–279 MAP antisera, BETO‐8039, BETO‐8040, and BETO‐8041, at dilutions of 10^?3, recognized both the rat GH‐BP_263–279 MAP and recombinant mouse GH‐BP with ED₅₀s within a range of 5–10 fmol, but did not cross‐react with BSA in dot blot analyses. BETO‐8041 antisera (10^?3 dilution) recognized GH‐BPs of rat serum and liver having M_rs ranging from 35 to 130 kDa, but did not recognize full‐length rat GH‐Rs. The antisera also detected recombinant mouse GH‐BPs. In summary, the tetravalent rat GH‐BP_263–279 MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C‐termini of both rat and mouse GH‐BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH‐BPs. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of a highly fluorescent N,N‐dimethyl benzylamine–palladium(II) curcuminate complex and its application for determination of trace amounts of water in organic solvents

In this work a new highly fluorescent N,N‐dimethyl benzylamine–palladium(II) yu complex was synthesized by the reaction of [Pd₂{(C,N–C₆H₄CH₂N(CH₃)₂}₂(μ‐OAc)]₂] with curcumin. The structure of the synthesized complex was characterized using Fourier transform infra‐red (FT‐IR) spectroscopy, ¹H nuclear magnetic resonance spectroscopy, and elemental analysis. Fluorescence quantum yield (Φ_F) values of the synthesized complex in dimethyl sulfoxide (DMSO), acetonitrile, ethanol, and methanol were 0.160, 0.104, 0.068, and 0.061, respectively. The fluorescence signal of the complex in the organic solvents was very sensitive to the water content of the organic solvent. The quenching effect of water was used to determine trace amounts of water in the heteroatom‐containing organic solvents (ethanol, methanol, acetonitrile) and redox‐active solvents (DMSO). The linear ranges for determination of water (v/v %) in ethanol, DMSO and acetonitrile were found to be 0.03–14.5, 0.08–13.8, and 0.07–18.8, respectively. Two linear ranges were found for determination of water (v/v %) in methanol (0.1–1.2 and 4.7–25.0). Detection limit (DL) values were calculated to be 0.001, 0.05, 0.004, and 0.01 (v/v %) in ethanol, methanol, acetonitrile, and DMSO, respectively. The proposed method overcomes the problems of the standard Karl Fischer method for determination of water in DMSO. In addition, it gave the best DL value for determination of water in ethanol compared with all published papers to date.     

Revealing the dimer dissociation and existence of a folded monomer of the mature HIV‐2 protease

Purification and in vitro protein‐folding schemes were developed to produce monodisperse samples of the mature wild‐type HIV‐2 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2_E37K (elevated K_d ～20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a T_m of 60.5 for PR2 as compared with 65.7°C for HIV‐1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the T_m of PR2 increases by 14.8°C in the presence of DRV as compared with 22.4°C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2_T26A) or a deletion of the C‐terminal residues 96–99 (PR2_1–95), drastically increase the K_d (>10⁵‐fold). PR2_T26A and PR2_1–95 consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size‐exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild‐type PR2 and its active‐site mutant PR2_D25N are folded dimers. Addition of twofold excess active‐site inhibitor promotes dimerization of PR2_T26A but not of PR2_1–95, indicating that subunit interactions involving the C‐terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.     

Lipopolysaccharide‐bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad‐spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram‐negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram‐negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α‐helical structure in a solution containing LPS. For NMR experiments, we expressed ¹⁵N‐labeled and ¹³C‐labeled CP1 in bacterial cells and successfully assigned almost all backbone and side‐chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr‐NOE) experiments in LPS. We performed ¹⁵N‐edited and ¹³C‐edited Tr‐NOE spectroscopy for CP1 bound to LPS. Tr‐NOE peaks were observed at the only C‐terminal region of CP1 in LPS. The results of structure calculation indicated that the C‐terminal region (Lys15–Gly29) formed the well‐defined α‐helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational studies of a bicyclic,lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–As p¹⁷, Lys26–As p³⁰)]PTHrP(1–34)NH₂, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly¹² and Arg¹⁹. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

What stabilizes the 314‐helix in β3‐peptides? A conformational analysis using molecular simulation

β‐Peptides are analogs of natural α‐peptides and form a variety of remarkably stable structures. Having an additional carbon atom in the backbone of each residue, their folded conformation is not only influenced by the side‐chain sequence but also and foremost by their substitution pattern. The precise mechanism by which the side chains interact with the backbone is, however, hitherto not completely known. To unravel the various effects by which the side chains influence the backbone conformation, we quantify to which extent the dihedral angles of a β³‐substited peptide with an additional methyl group on the central C_α‐atom can be regarded as independent degrees of freedom and analyze the distributions of these dihedral angles. We also selectively capture the steric effect of substituents on the C_α‐ and C_β‐atoms of the central residue by alchemically changing them into dummy atoms, which have no nonbonded interactions. We find that the folded state of the β³‐peptide is primarily stabilized by a steric exclusion of large parts of the unfolded state (entropic effect) and only subsequently by mutual dependence of the ψ‐dihedral angles (enthalpic effect). The folded state of β‐peptides is stabilized by a different mechanism than that of α‐peptides. Proteins 2010. © 2010 Wiley‐Liss, Inc.