Interaction of Halictine-Related Antimicrobial Peptides with Membrane Models.

We have investigated structural changes of peptides related to antimicrobial peptide Halictine-1 (HAL-1) induced by interaction with various membrane-mimicking models with the aim to identify a mechanism of the peptide mode of action and to find a correlation between changes of primary/secondary structure and biological activity. Modifications in the HAL-1 amino acid sequence at particular positions, causing an increase of amphipathicity (Arg/Lys exchange), restricted mobility (insertion of Pro) and consequent changes in antimicrobial and hemolytic activity, led to different behavior towards model membranes. Secondary structure changes induced by peptide-membrane interaction were studied by circular dichroism, infrared spectroscopy, and fluorescence spectroscopy. The experimental results were complemented by molecular dynamics calculations. An α-helical structure has been found to be necessary but not completely sufficient for the HAL-1 peptides antimicrobial action. The role of alternative conformations (such as ß-sheet, PPII or 310-helix) also seems to be important. A mechanism of the peptide mode of action probably involves formation of peptide assemblies (possibly membrane pores), which disrupt bacterial membrane and, consequently, allow membrane penetration.

Electronic circular dichroism of the chiral rigid tricyclic dilactam with nonplanar tertiary amide groups.

Electronic circular dichroism (ECD) of the spirocyclic dilactam 5,8-diazatricyclo[6,3,0,0(1,5)]undecane-4,9-dione has been measured in the extended wavelength range (170-260 nm) utilizing far-UV CD instrumentation including synchrotron radiation light source. The data of this model of two nonplanar tertiary amide groups interacting within the rigid chiral environment provided new information particularly about the shorter wavelength π-π* transition region below 190 nm. The interpretation using TDDFT calculations confirmed that effects of amide nonplanarity follow our previous observations on monolactams as far as amide n-π* transitions are concerned. ECD band in the n-π* transition region of the nonplanar diamide exhibits an identical bathochromic shift and its sign remains tied to the sense of nonplanar deformation in the same way. As far as n-π* transitions are concerned amide nonplanarity acts as a local phenomenon independently reflecting sum properties of single amide groups. On the other hand, CD bands associated with π-π* transitions (found between ∼170 to 210 nm) form an exciton-like couplet with the sign pattern determined by mutual orientation of the associated electric transition moments. This sign pattern follows predictions pertaining to a coupled oscillator. The influence of amide nonplanarity on π-π* transitions is only minor and concentrates into the shorter wavelength lobe of the π-π* couplet. The detailed analysis of experimental ECD with the aid of TDDFT calculations shows that there is only little interaction between effects of inherent chirality caused by nonplanarity of amide groups and amide-amide coupling. Consequently these two effects can be studied nearly independently using ECD. In addition, the calculations indicate that participation of other type of transitions (n-σ*, π-σ* or Rydberg type transitions) is only minor and is concentrated below 180 nm.

Nonplanar tertiary amides in rigid chiral tricyclic dilactams. Peptide group distortions and vibrational optical activity.

We investigate amide nonplanarity in vibrational optical activity (VOA) spectra of tricyclic spirodilactams 5,8-diazatricyclo[6,3,0,0(1,5)]undecan-4,9-dione (I) and its 6,6',7,7'-tetradeuterio derivative (II). These rigid molecules constrain amide groups to nonplanar geometries with twisted pyramidal arrangements of bonds to amide nitrogen atoms. We have collected a full range vibrational circular dichroism (VCD) and Raman optical activity (ROA) spectra including signals of C-H and C-D stretching vibrations. We report normal-mode analysis and a comparison of calculated to experimental VCD and ROA. The data provide band-to-band assignment and offer a possibility to evaluate roles of constrained nonplanar tertiary amide groups and rigid chiral skeletons. Nonplanarity shows as single-signed VCD and ROA amide I signals, prevailing the couplets expected to arise from the amide-amide interaction. Amide-amide coupling dominates amide II (mainly C'-N stretching, modified in tertiary amides by the absence of a N-H bond) transitions (strong couplet in VCD, no significant ROA) probably due to the close proximity of amide nitrogen atoms. At lower wavenumbers, ROA spectra exhibit another likely manifestation of amide nonplanarity, showing signals of amide V (δ(oop)(N-C) at ~570 cm(-1)) and amide VI (δ(oop)(C'═O) at ~700 cm(-1) and ~650 cm(-1)) vibrations.

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.

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.

Coiled coil peptides as universal linkers for the attachment of recombinant proteins to polymer therapeutics.

We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.

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.

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 cyclodextrin's 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.

Vibrational and electronic optical activity of the chiral disulphide group: implications for disulphide bridge conformation.

Using dihydrogendisulphide (H(2)S(2)), dimethyl- ((CH(3))(2)S(2)), and diethyldisulphide ((CH(3)CH(2))(2)S(2))as model molecules, theoretical ECD, VCD, and ROA spectra of nonplanar disulphides were calculated by DFT methods. Most of the calculated electronic and vibrational chiroptical features suffer an equivocal relation between calculatedsigns of ECD, VCD, or ROA and the sense of disulphide nonplanarity as noted earlier for low-lying ECD bands. This is a consequence of local C(2) symmetry of a disulphide group causing most electronic and vibrational transitions to occur as pairs falling to alternative A, B symmetry species, which become degenerate and switch their succession (and consequently the observed chiroptical sign pattern) at the energetically most favorable perpendicular conformation. According to present calculations, the key to resolving this ambiguity may involve the S-S stretching vibrational mode at approximately 500 cm(-1). The relation of signs of the relevant VCD and ROA features to sense of disulphide chirality seems simpler and less ambiguous. The right-handed arrangement of the S-S group (0 < chi(S-S) < 180 degrees) results in mostly negative VCD signals. Although relation to ROA still suffers some ambiguity, it gets clearer along the series H(2)S(2)-(CH(3))(2)S(2)-(CH(3)CH(2))(2)S(2). ROA is also attractive for the analysis of disulphide-containing peptides and proteins, because applying it to aqueous solutions is not problematic.

Comparison of vibrational circular dichroism instruments: development of a new dispersive VCD.

A dispersive vibrational circular dichroism (VCD) instrument has been designed and optimized for the measurement of mid-infrared (MIR) bands such as the amide I and amide II vibrational modes of peptides and proteins. The major design considerations were to construct a compact VCD instrument for biological molecules, to increase signal-to-noise (S/N) ratio, to simultaneously collect and digitize the sample transmission and polarization modulation signals, and to digitally ratio them to yield a VCD spectrum. These were realized by assembling new components using design factors adapted from previous VCD instruments. A collection of spectra for peptides and proteins having different dominant secondary structures (alpha-helix, beta-sheet, and random coil) measured for identical samples under the same conditions showed that the new instrument had substantially improved S/N as compared with our previous dispersive VCD instrument. These instruments both provide protein VCD for the amide I that are comparable to or somewhat better than those measurable with commercial Fourier transform (FT) VCD instruments if just the amide I band in the spectra is obtained at modest resolution (8 cm(-1)) with the same total data collection time on each type of instrument.

Spectroscopic properties of the nonplanar amide group: a computational study.

Experimental studies suggest that amide bond may significantly deviate from planar arrangement even in linear peptides and proteins. In order to find out the extent to which such deviation may influence principal amide spectroscopic properties, we conducted a computational study of nonplanar N-methylacetamide (NMA) conformers. Vibrational absorption, Raman, and electronic spectra including optical activity were simulated with ab initio and density functional theory (DFT) methods. According to the results, small nonplanarity deviations may be detectable by nonpolarized spectroscopic techniques, albeit as subtle spectral changes. The optical activity methods, such as the vibrational circular dichroism (VCD), Raman optical activity (ROA), and electronic circular dichroism (CD, ECD), provide enhanced information about the amide nonplanarity, because planar amide is not optically active (chiral). For VCD, however, the inherently chiral contribution in most peptides and proteins most probably provides very weak signal in comparison with other contributions, such as the dipolar coupling. For the electronic CD, the nonplanarity contribution is relatively big and causes a strong CD couplet in the n-pi* absorption region accompanied by a red frequency shift. The pi-pi* CD region is relatively unaffected. The ROA spectroscopy appears most promising for the nonplanarity detection and the inherent chiral signal may dominate entire spectral parts. The amide I and III vibrational ROA bands are most challenging experimentally because of their relatively weak coupling to other peptide vibrations.

The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase.

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of alpha-helical conformation, as measured by circular dichroism. A similar effect was observed when a His(6)-tag was linked to the C-terminus of Sapp1p or its precursor.

Determination of quality of pyridoxal-5'-phosphate enzyme preparations by spectroscopic methods.

The present study evaluates purified aspartate transaminase (AST, EC 2.6.1.1) preparations from three commercial sources. The enzyme molecule contains pyridoxal-5'-phosphate coenzyme (PLP), which provides AST characteristic absorption spectra in the wavelength range of 300-500 nm. The coenzyme bound in the active site also shows circular dichroism (CD) spectra in the same range. Besides, AST like other proteins may be modified in vitro or in vivo by reactions with other molecules, e.g. reactive sugars, and may form fluorescent products (advanced glycation end products, AGE). Spectroscopic methods were used to assess the quality of AST preparations from three different sources, Serva, Roche, and Sigma. Absorption spectra showed that the peak 360 nm characteristic of the active PLP form of AST prevailed in the Serva and Sigma preparations, while 330 nm was the major peak in the Roche preparation. CD spectra demonstrated the major maximum at 360 nm in the Serva and Roche samples, thus suggesting the predominance of the active PLP form in both preparations. The Sigma sample showed a CD profile less characteristic of AST. Fluorescence measurements revealed formation of AGE in the case of the Roche preparation, while fluorescence of the other two preparations was low. In general, the Serva sample presented the most convenient properties of purified AST among the preparations tested. The results will be used for the selection of a commercial enzyme preparation applicable in our future spectroscopic studies of glycation of AST as a model protein and in our research of the influence of antioxidants on this process.

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 K(d) 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 approximately 20 degrees 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 alpha-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 alpha-helical regions of the G-domain by Ecdomain 2.