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Ž .A direct transfer of Cartesian molecular force fields FF and electric property tensors is tested on model systems and compared to transfer in internal coordinates with an aim to improve simulation of vibrational spectra for larger molecules. This Cartesian transformation can be implemented easily and offers greater flexibility in practical computations. It can be also applied for transfer of anharmonic derivatives. The results for model calculations of the force field and vibrational frequencies for N-methylacetamide show that our method removes errors associated with numerical artifacts caused by nonlinearity of the otherwise required Cartesian to internal coordinate transformation. For determination of IR absorption and vibrational circular dichroism intensities, atomic polar and axial tensors were also transferred in the Cartesian representation. For the latter, which are dependent upon the magnetic dipole operator, a distributed origin gauge is used to avoid an origin dependence. Comparison of the results of transferring ab initio FF and intensity parameters from an amide dimer fragment onto a tripeptide with those from a conventionally determined tripeptide FF document some limitations of the transfer method and its possible applications in the vibrational spectroscopy. Finally, application to determination of the FF and spectra for helical heptapeptide are presented and compared to experimental results.

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.

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.

Central cyclic part of the hinge peptide (a parallel dimer of the pentapeptide Boc-Cys-Pro-Pro-Cys-Pro-NHCH3 with two disulfide bonds) derived from the sequence of human IgG1 is a rather rigid structure having predominantly polyproline II helical conformation as shown by vibrational circular dichroism spectra. It exhibits significant Raman optical activity (ROA) signal due to vibrations associated with the disulfide bridges. We report positive ROA for the S-S stretching vibration at 510 cm-1 and for the C-S stretching vibration at 655 cm-1. These signals can provide means to assess the conformation of disulfide bridges in proteins, otherwise difficult to investigate.

Nuclear magnetic resonance (NMR) and Raman optical activity (ROA) spectra of the cyclic dipeptide were measured and analyzed with respect to their ability to sense molecular structure and conformation. Data obtained by both techniques were simulated using ab initio quantum mechanical computations. Calculated chemical shifts, hydrogen-hydrogen spin-spin coupling constants and ROA intensities agreed well with the experimental values. The spin-spin NMR coupling constants were found to be most suitable for estimating of the conformational ratio. The ROA intensities provided additional information about the absolute configuration. The relation of the NMR chemical shifts to molecular structure was obscured by the solvent effect. The experimental results and calculated relative conformer energies suggest that equilibrium of three conformations takes place in the solution at the room temperature with a prevalence (∼80%) of the conformation present in the crystalline state.

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.

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.