Structure of a simplified ß-hairpin and its ATP complex.

The capacity of three designed duodecamer peptides with the low diversity sequence: H1ϕ2I3K4I5D6G7K8ϕ9I10K11H12 where ϕ is His, Phe or Trp, to adopt a ß-hairpin conformation was studied using NMR spectroscopy. Whereas KIAßH, the variant with His at positions two and nine, is disordered, KIAßF, the peptide with Phe at these positions, adopts a small population of ß-hairpin. A high population of ß-hairpin structure was detected for KIAßW, the variant with Trp. Utilizing NMR data, the structure of KIAßW was solved and it reveals a ß-hairpin stabilized by hydrophobic interactions between Ile residues on one face and Trp-Trp and cation-π interactions on the opposite face. Upon adding ATP, these peptides show chemical shift changes indicative of ATP binding. The binding of ATP to KIAßW shows a KD ≈ 20 µM at pH 5, 5 °C and has a 1:1 stoichiometry. The KIAßW-ATP complex was determined using NMR spectroscopy and reveals the adenine ring sandwiched between the two Trp indole rings and that ATP binding induces important conformational changes in His1, Trp2, Lys4, Trp9 and Lys11 in the ß-hairpin. The implications of these results for the hypothetic presence of ß-hairpins and amyloids alongside RNAs on the prebiotic Earth are discussed.

An Arg-rich putative prebiotic protein is as stable as its Lys-rich variant.

An Arg-rich peptide called RIA7; sequence ac-ARAAAAAIRAIAAIIRAGGY-am, tetramerizes to form a well folded, four helix X-bundle protein. The Arg side chains are solvent exposed and the hydrophobic core is composed of the side chains from some Alas, all the Iles and the C-terminal Tyr. Since Gly, Ala and Ile, and in lesser amounts Arg and Tyr have been reported to form under putative prebiotic Earth conditions, it is plausible that RIA7-like peptides might have formed on the primitive Earth and interacted with RNAs. The interaction of RIA7 with two RNAs was tested and the formation of insoluble aggregates was observed. These results contrast with previous studies of a Lys-rich variant, called KIA7, which promotes the cleavage of RNAs. Their close structural similarity makes RIA7 and KIA7 an excellent system to compare the relative contributions of Arg and Lys to protein conformational stability. NMR-monitored hydrogen/deuterium exchange measurements and CD-monitored thermal denaturation experiments performed at different peptide and salt concentrations reveal that the conformational stabilities of RIA7 and KIA7 are practically the same. This finding has relevance for protein engineering as Lys is frequently replaced by Arg to improve ligand binding and membrane association and penetration.

Conformation specificity and arene binding in a peptide composed only of Lys, Ile, Ala and Gly.

The first life on Earth is believed to have been based on RNA, but might have taken advantage of amino acids and short peptides which form readily under conditions like those of the primitive Earth. We have shown that simple peptides adopt specifically folded four-helix bundle structures that can recognize and cleave RNA. Here, to explore the limits of conformational specificity, we characterize a simpler peptide composed of just Lys, Ile, Ala, and Gly called KIA7I. Using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations, we find kinks in the helices of KIA7I and multiple C-terminal conformations. These results suggest that the C-terminal Ile residue does not completely occupy the hydrophobic pocket that is filled by aromatic side-chains in well-folded KIA7 variants. The capacity of arenes to fill this cavity was tested. Using NMR, we show that benzene and phenol can bind KIA7I, but do not bind the well-folded variant KIA7W or hen egg white lysozyme. Benzene also binds Aß(1-40), a mostly disordered polypeptide implicated in Alzheimer's disease. 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence is further enhanced in the presence of both KIA7I and arenes relative to KIA7I alone. This ANS fluorescence enhancement is stronger for smaller and less polar arenes and less ordered KIA variants. These results suggest that arenes are not confined to the pocket, but penetrate and loosen the hydrophobic core of KIA7I.

In vitro evaluation of the antielastase activity of polycyclic ß-lactams.

A series of bi- and tricyclic ß-lactam compounds was synthesized and evaluated as inhibitors of cleavage of synthetic substrates in vitro by the serine proteases Human Leukocyte Elastase (HLE), Human Leukocyte Proteinase 3 (HLPR3) and Porcine Pancreatic Elastase (PPE). The obtained results have permitted us to describe a homobenzocarbacephem compound as HLE and HLPR3 inhibitor, to observe the positive effect that the styryl group exerts on the HLE inhibitory activity in polycyclic ß-lactam compounds and to conclude that the hydroxyl function decreases the HLE inhibitory activity or rules it out completely.

Carbodiimide EDC induces cross-links that stabilize RNase A C-dimer against dissociation: EDC adducts can affect protein net charge, conformation, and activity.

RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDC-treated RNase A monomer were found to carry an increased number of positive charges (about 6 ± 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH(2) monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.