Petr Maloň

Lucifensin, a novel insect defensin of medicinal maggots: synthesis and structural study.

Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram‐positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16–Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N‐terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α‐helix‐promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.

Liquid chromatographic resolution of enantiomers of deltahedral carborane and metallaborane derivatives

Abstract A successful liquid chromatographic separation of twelve enantiomeric pairs of zwitterionic eleven-vertex nido -carboranes of the type L-R-7,8-C 2 B 9 H 10 (L = Me 2 S, Me 2 SCH 2 , C 6 H 5 N, C 6 H 5 NCH 2 ; R = H, CH 3 , C 6 H 5 , Me = methyl) and of the 4-MeS-3-C 5 H 5 -1,2,3-C 2 CoB 9 H 10 mixed sandwich complex on β-cyclodextrin (CD) chiral stationary phases (CSP) in aqueous-organic mobile phases is described. A comparison of two β-CD CSP columns. Tessek β-CD and Astec Cyclobond I, differing in enantioselectivity and resolution of individual compounds (to some extent), is presented together with a study of the factors controlling retention and enantiomeric resolution.

Thermostability of multidomain proteins: Elongation factors EF‐Tu from Escherichia coli and Bacillus stearothermophilus and their chimeric forms

Recombinant mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) elongation factors EF‐Tus, their isolated G‐domains, and six chimeric EF‐Tus composed of domains of either EF‐Tu were prepared, and their GDP/GTP binding activities and thermostability were characterized. BstEF‐Tu and BstG‐domain bound GDP and GTP with affinities in nanomolar and submicromolar ranges, respectively, fully comparable with those of EcEF‐Tu. In contrast, the EcG‐domain bound the nucleotides with much lower, micromolar affinities. The exchange of domains 2 and 3 had essentially no effect on the GDP‐binding activity; all complexes of chimeric EF‐Tus with GDP retained Kd values in the nanomolar range. The final thermostability level of either EF‐Tu was the result of a cooperative interaction between the G‐domains and domains 2 + 3. The G‐domains set up a “basic” level of the thermostability, which was ∼20°C higher with the BstG‐domain than with the EcG‐domain. This correlated with the growth temperature optimum difference of both bacteria and two distinct thermostabilization features of the BstG‐domain: an increase of charged residues at the expense of polar uncharged residues (CvP bias), and a decrease in the nonpolar solvent‐accessible surface area. Domains 2 + 3 contributed by further stabilization of α‐helical regions and, in turn, the functions of the G‐domains to the level of the respective growth temperature optima. Their contributions were similar irrespective of their origin but, with Ecdomains 2 + 3, dependent on the guanine nucleotide binding state. It was lower in the GTP conformation, and the mechanism involved the destabilization of the α‐helical regions of the G‐domain by Ecdomain 2.

Models of pigment-protein interactions in photosynthetic systems: Tetraphenylporphyrin complexes with polycationic sequential polypeptides. Absorption, circular dichroism and fluorescence properties

Abstract Absorption and circular dichroism spectra and steady state and time-resolved fluorescence of complexes of 5,10,15,20-tetrakis (4-carboxyphenyl)porphyrin with sequential helical polypeptides (L-Lys-L-Ala) n , (L-Lys-L-Ala-L-Ala) n , and (L-Lys-L-Ala-L-Ala-L-Ala) n are studied. Changes of electronic transition energies, induction of optical activity and two-component fluorescence decay with components t 1 = 2–3 ns and t 2 = 180 ps characterize the complexed pigment. Dependence of these changes on relative number of lysine amino acids in polypeptide primary structures was observed and is discussed in terms of perturbational influence of polypeptide molecular field on mixing of porphyrin electronic states.

Theoretical simulation of a polarization modulator based on mechanical rotation of a polarizing element

The properties of three alternative designs for a polarization modulator of potential use for the measurement of vibrational circular dichroism (VCD) are evaluated and compared by use of Mueller calculus. The analysis shows that the combination of a fixed polarizer plus either a photoelastic modulator or a rotating quarter-wave plate possesses nearly the same capability for generation of time-varying, circularly polarized light. However, a modulator composed of a rotating polarizer plus a fixed birefringent plate entails considerable theoretical and experimental difficulties for use in the measurement of VCD spectra. While VCD spectra obtained with the rotating devices can be calibrated in the same manner as spectra obtained with a photoelastic modulator, Mueller analysis shows that the form of the resultant calibration signal will have a different shape. The relevant expressions for VCD and linear dichroism as well as the calibration signals are presented, and consequences for practical realization of these experiments are discussed.

Lasiocepsin, a novel cyclic antimicrobial peptide from the venom of eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae).

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8–Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17–Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.

Disulfide chromophore and its optical activity

The compounds I-IV derived from α-D-cyclodextrin moiety by bridging and/or interconnecting with various patterns of disulfide bonds were chosen as models for the spectroscopic study of conformation of the disulfide bridge. The energy gap between the disulfide and cyclodextrins electronic transitions allows us to investigate absorption and electronic circular dichroism spectra without disturbing spectral overlaps with amides or aromatic amino acids in peptides or proteins. Raman optical activity (ROA) spectra were measured and the bands due to S-S and C-S stretching motion identified. Comparison with the quantum mechanical calculations of simple models indicates that sense of disulfide twist follows sign of the measured S-S ROA band.

Axially chiral bis(α-amino acid)s and dioxopiperazines. Synthesis and configurational assignment

Abstract Conversion of the easily accessible racemic bis(amino acid)s cis- and trans-2a into the (S)-enantiomers via the corresponding Schiff bases with (1S,2S,5S)-2-hydroxy-3-pinanone is described. Condensation of the N-Boc-protected bis(amino acid)s with esters of the corresponding axially chiral amino acids (R)- and (S)- 1b afforded tetraspiro-substituted bis(dioxopiperazine)s 7 representing a rare case of primary helical topology. Absolute configuration of the products was assigned by NMR and CD spectra and confirmed by X-ray analysis of the Schiff base (S)-cis- 3 .

Antimicrobial Peptide from the Eusocial Bee Halictus sexcinctus Interacting with Model Membranes

Halictine-1 (Hal-1)—a linear antibacterial dodecapeptide isolated from the venom of the eusocial bee Halictus sexcinctus—has been subjected to a detailed spectroscopic study including circular dichroism, fluorescence, and vibrational spectroscopy. We investigated Hal-1 ability to adopt an amphipathic α-helical structure upon interaction with model lipid-based bacterial membranes (phosphatidylcholine/phosphatidylglycerol-based large unilamellar vesicles and sodium dodecylsulfate micelles) and helix inducing components (trifluoroethanol). It was found that Hal-1 responds sensitively to the composition of the membrane model and to the peptide/lipid ratio. The amphipathic nature of the helical Hal-1 seems to favour flat charged surfaces of the model lipid particles over the nondirectional interaction with trifluoroethanol. Increasing fraction of polyproline II type conformation was detected at low peptide/lipid ratios.

Electronic and Vibrational Optical Activity of Several Peptides Related to Neurohypophyseal Hormones: Disulfide Group Conformation

Electronic and vibrational optical activity of the set of neurohypophyseal hormones and their analogs was investigated to clarify the S-S bond solution conformation. The selected compounds include oxytocin (I), lysine vasopressin (II), arginine vasopressin (III), and their analogs (IV-IX), differing widely in their pharmacological properties. We have extended the already known electronic circular dichroism data by new information provided by vibrational circular dichroism (VCD) and Raman optical activity (ROA). The use of VCD brought additional details on three-dimensional structure of the chain reversal in the ring moiety and on its left handedness. Furthermore, Raman scattering and ROA allowed us to deduce the sense of the disulfide bond torsion.