N-Me-Phe-OH

Forty-five crystals of complexes between proteins and RNA molecules from the Protein Data Bank have been statistically surveyed for the number of contacts between RNA components (phosphate, ribose and the four bases) and amino acid side chains. Three groups of complexes were defined: the tRNA synthetases; the ribosomal complexes; and a third group containing a variety of complexes. The types of atomic contacts were a priori classified into ionic, neutral H-bond, C-H...O H-bond, or van der Waals interaction. All the contacts were organized into a relational database which allows for statistical analysis. The main conclusions are the following: (i) in all three groups of complexes, the most preferred amino acids (Arg, Asn, Ser, Lys) and the less preferred ones (Ala, Ile, Leu, Val) are the same; Trp and Cys are rarely observed (respectively 15 and 5 amino acids in the ensemble of interfaces); (ii) of the total number of amino acids located at the interfaces 22% are hydrophobic, 40% charged (positive 32%, negative 8%), 30% polar and 8% are Gly; (iii) in ribosomal complexes, phosphate is preferred over ribose, which is preferred over the bases, but there is no significant preference in the other two groups; (iv) there is no significant prevalence of a base type at protein-RNA interfaces, but specifically Arg and Lys display a preference for phosphate over ribose and bases; Pro and Asn prefer bases over ribose and phosphate; Met, Phe and Tyr prefer ribose over phosphate and bases. Further, Ile, Pro, Ser prefer A over the others; Leu prefers C; Asp and Gly prefer G; and Asn prefers U. Considering the contact types, the following conclusions could be drawn: (i) 23% of the contacts are via potential H-bonds (including CH...O H-bonds and ionic interactions), 72% belong to van der Waals interactions and 5% are considered as short contacts; (ii) of all potential H-bonds, 54% are standard, 33% are of the C-H...O type and 13% are ionic; (iii) the Watson-Crick sites of G, O6(G) and principally N2(G) and the hydroxyl group O2' is more often involved in H-bonds than expected; the protein main chain is involved in 32% and the side chains in 68% of the H-bonds; considering the neutral and ionic H-bonds, the following couples are more frequent than expected-base A-Ser, base G-Asp/Glu, base U-Asn. The RNA CH groups interact preferentially with oxygen atoms (62% on the main chain and 19% on the side chains); (iv) the bases are involved in 38% of all H-bonds and more than 26% of the H-bonds have the H donor group on the RNA; (v) the atom O2' is involved in 21% of all H-bonds, a number greater than expected; (vi) amino acids less frequently in direct contact with RNA components interact frequently via their main chain atoms through water molecules with RNA atoms; in contrast, those frequently observed in direct contact, except Ser, use instead their side chain atoms for water bridging interactions.

Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze the NADPH-dependent oxidation of a large number of sulfur-, selenium-, and nitrogen-containing compounds. Five active isoforms (FMO1-5) have been identified and shown to be differently expressed in various mammalian tissues. Previous work from this laboratory has shown l-methionine to be S-oxidized by rat, rabbit and human FMO1-4, with FMO3 exhibiting the highest stereoselectivity for the formation of the d-diastereomer of methionine sulfoxide. In this report, we describe new studies aimed at determining if N-acetyl-l-methionine and peptides containing l-methionine can be substrates for FMOs. Experiments were carried out using either rabbit liver microsomes or human cDNA-expressed FMOs. The results show that while N-acetyl-l-methionine and peptides with a modified methionine amino group may not function as substrates for FMOs, peptides containing a free N-terminal methionine may act as FMO substrates. With human cDNA-expressed FMO1, FMO3, and FMO5, both FMO1 and FMO3 exhibited activity with the active peptides whereas FMO5 was inactive. With FMO3, the activity measured with methionine was similar (1 mM) or higher (5 mM) than the activity measured with H-Met-Val-OH and H-Met-Phe-OH. With FMO1, H-Met-Phe-OH and methionine exhibited similar activities whereas activity with H-Met-Val-OH was much lower. Collectively, the results show that FMOs can oxidize peptides containing a free N-terminal methionine. Thus, the role of FMOs in the oxidation of methionine in larger peptides or proteins warrants further investigation.

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.