Zsuzsanna Majer

Synthesis and Conformational Analysis of Efrapeptins

The efrapeptin family of peptide antibiotics produced by the fungus Tolypocladium niveum, and the neo-efrapeptins from the fungus Geotrichum candidumare inhibitors of F(1)-ATPase with promising antitumor, antimalaria, and insecticidal activity. They are rich in C(α)-dialkyl amino acids (Aib, Iva, Acc) and contain one β-alanine and several pipecolic acid residues. The C-terminus bears an unusual heterocyclic cationic cap. The efrapeptins C-G and three analogues of efrapeptin C were synthesized using α-azido carboxylic acids as masked amino acid derivatives. All compounds display inhibitory activity toward F(1)-ATPase. The conformation in solution of the peptides was investigated with electronic CD spectroscopy, FT-IR spectroscopy, and VCD spectroscopy. All efrapeptins and most efrapeptin analogues were shown to adopt helical conformations in solution. In the case of efrapeptin C, VCD spectra proved that a 3(10)-helix prevails. In addition, efrapeptin C was conformationally studied in detail with NMR and molecular modeling. Besides NOE distance restraints, residual dipolar couplings (RDC) observed upon partial alignment with stretched PDMS gels were used for the conformational analysis and confirmed the 3(10)-helical conformation.

Is β-homo-proline a pseudo-γ-turn forming element of β-peptides? An IR and VCD spectroscopic study on Ac-β-HPro-NHMe in cryogenic matrices and solutions

In order to test the pseudo-γ-turn forming capability of β-homo-proline (β(3)-HPro) 2-[(2S)-1-acetylpyrrolidin-2-yl]-N-methylacetamide (Ac-β(3)-HPro-NHMe) was synthesized and its potential energy landscape was investigated by infrared (IR) and vibrational circular dichroism (VCD) spectroscopy combined with density functional calculations. Based upon a comparison between experimental and computed spectra three different pseudo-γ-turn-like trans conformers and a cis conformer were identified in low-temperature Ar and Kr matrices. The computations in agreement with the observations reveal that, in contrast to its α-Pro analogue, the room-temperature abundance of the cis conformer is significant, falling above 10% in the isolated phase. Furthermore, solution-phase vibrational spectra and computations show that the cis conformer is predominant in polar solvents. This result indicates that β(3)-HPro is significantly less apt to form pseudo-γ-turns when compared to the γ-turn forming tendency of α-proline. The present study also shows that the interpretation of solution-phase VCD spectra of flexible molecules should be done with extra caution.

Single-Molecule Experiments to Elucidate the Minimal Requirement for DNA Recognition by Transcription Factor Epitopes

Interactions between proteins and DNA are essential for the regulation of cellular processes in all living organisms. In this context, it is of special interest to investigate the sequence-specific molecular recognition between transcription factors and their cognate DNA sequences. As a model system, peptide and protein epitopes of the DNA-binding domain (DBD) of the transcription factor PhoB from Escherichia coli are analyzed with respect to DNA binding at the single-molecule level. Peptides representing the amphiphilic recognition helix of the PhoB DBD (amino acids 190-209) are chemically synthesized and C-terminally modified with a linker for atomic force microscopy-dynamic force spectroscopy experiments (AFM-DFS). For comparison, the entire PhoB DBD is overexpressed in E. coli and purified using an intein-mediated protein purification method. To facilitate immobilization for AFM-DFS experiments, an additional cysteine residue is ligated to the protein. Quantitative AFM-DFS analysis proves the specificity of the interaction and yields force-related properties and kinetic data, such as thermal dissociation rate constants. An alanine scan for strategic residues in both peptide and protein sequences is performed to reveal the contributions of single amino acid residues to the molecular-recognition process. Additionally, DNA binding is substantiated by electrophoretic mobility-shift experiments. Structural differences of the peptides, proteins, and DNA upon complex formation are analyzed by circular dichroism spectroscopy. This combination of techniques eventually provides a concise picture of the contribution of epitopes or single amino acids in PhoB to DNA binding.

Computational structure-activity study directs synthesis of novel antitumor enkephalin analogs

The capability of a Support Vector Machines QSAR model to predict the antiproliferative ability of small peptides was evaluated by screening a virtual library of enkephalin-like analogs modified by incorporation of the (R,S)-(1-adamantyl)glycine (Aaa) residue. From an initial set of 390 compounds, the peptides, Tyr-Aaa-Gly-Phe-Met (2), Tyr-Aaa-Gly-Phe-Phe (3), Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5) were selected, synthesized and their antitumor activity was tested and compared to that of Met-enkephalin (1). The antiproliferative activity correlated with the computational prediction and with the foldamer-forming ability of the studied peptides. The most active compounds were the hydrophobic peptides, Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5), having a greater propensity to adopt folded structures than the other peptides.

The structure of horseradish peroxidase C characterized as a molten globule state after Ca2+ depletion

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca(2+) ions. Earlier studies suggested a critical role of one of the bound Ca(2+) ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca(2+)-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca(2+)-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca(2+), perturbs a H-bonding network connecting this Ca(2+) to the heme crevice that involves the distal water. The measured effects of Ca(2)(+) depletion can be interpreted as supporting a structural role for the distal Ca(2+) and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.

VCD studies on cyclic peptides assembled from L-α-amino acids and a trans-2-aminocyclopentane- or trans-2-aminocyclohexane carboxylic acid

The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans‐2‐aminocyclohexane carboxylic acid (Achc) or trans‐2‐aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three‐dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6‐31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR‐based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides. Copyright

Conformational properties of secondary amino acids: replacement of pipecolic acid by N-methyl-l-alanine in efrapeptin C.

The efrapeptins, a family of naturally occurring peptides with inhibitory activities against ATPases, contain several α,α‐disubstituted α‐amino acids such as α‐aminoisobutyric acid (Aib) or isovaline (Iva) besides pipecolic acid (Pip), β‐Ala, Leu, Gly, and a C‐terminal heterocyclic residue. Secondary α‐amino acids such as proline are known to stabilize discrete conformations in peptides. A similar influence is ascribed to N‐alkyl α‐amino acids. We synthesized two efrapeptin C analogs with replacement of Pip by N‐methyl‐L‐alanine (MeAla) using a combination of solid‐ and solution‐phase techniques in a fragment‐condensation strategy to compare the conformational bias of both secondary amino acids. The solution conformation was investigated by vibrational circular dichroism (VCD) to probe whether the analogs adopt a 310‐helical conformation. The MeAla‐containing analogs [MeAla1,3]efrapeptin C and [MeAla1,3,11]efrapeptin C inhibit ATP hydrolysis by the A3B3 complex of A1A0‐ATP synthase from Methanosarcina mazei Gö1.

Coacervation and CD and FT-IR Spectroscopic Studies of Elastin-Derived Polypentapeptide and Its Analogs

Elastin imparts elasticity to elastic tissues such as arterial walls, lungs, ligaments, and skin. In vivo, tropoelastin, a precursor of insoluble elastin, self-associates with appropriate alignment in extracellular spaces, followed by cross-linking to form elastin. The self-association of tropoelastin molecules is the most significant step in the process of elastin biosynthesis, termed coacervation in vitro. The polypentapeptide (VPGVG)n, one of the repeating peptide sequences in the elastin molecule, is the most striking primary structural feature of elastin. In order to clarify the molecular mechanism of coacervation, we synthesized the polypentapeptide (V1P2G3V4G5)n and its three analogs (Figure 1) — (V1P2G3V4)n in which Gly5 is deleted, (V1P2G3)n in which both Gly5 and Val4 are deleted, and (P2G3V4G5)n in which Val1 is deleted. The coacervation properties and conformational analyses of these polypeptides were investigated.