Thioether Cross-links Research Articles

Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestgamma-subunit of the alphabetagamma heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the alpha- and gamma-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The alpha- and beta-subunits were both detected in the periplasm of the mutant strain, whereas the gamma-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the gamma-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of gamma-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.

Active Site Aspartate Residues Are Critical for Tryptophan Tryptophylquinone Biogenesis in Methylamine Dehydrogenase

The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue betaTrp57 and covalent cross-linking of betaTrp57 to betaTrp108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues betaAsp76 and betaAsp32 are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative betaD76N and betaD32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. betaD76N MADH possessed the six disulfides but had no oxygen incorporated into betaTrp57 and was completely inactive. The betaD32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into betaTrp57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the betaD32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the Km value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.

Synthesis and activity of dimeric bradykinin antagonists containing diaminodicarboxylic acid bridge residues.

Enhancement of a ligand's interaction with a receptor through presenting the ligand in multimeric form is a topic of general interest. Thus dimerization of single-chain bradykinin antagonist peptides has previously been shown to be beneficial in terms of potency and duration of action. While crosslinking polypeptides at terminal positions using suitable dicarboxylic acids and diamines is comparatively straight-forward synthetically, internal dimerizations are usually achieved through oxidation or double S-alkylations of cysteine residues, resulting in metabolically unfavourable disulphide and thioether cross-links. Using suitably modified standard solid-phase peptide synthesis protocols, dimeric bradykinin antagonist peptides [H-(D-Arg)-Arg-Pro-Hyp-Gly-Phe]2-X-[(D-Phe)-Leu-Arg-OH]2 were synthesized where X corresponds to a L,L-2,7-diaminosuberic or L,L-2,9-diaminosebacic acid residue, respectively. The biological activity of these peptides was comparable to that of conventional dimeric bradykinin antagonists cross-linked through cystine or bis(succinimido)alkyl bridges.

Structure-based design of high affinity streptavidin binding cyclic peptide ligands containing thioether crosslinks.

High affinity cyclic thioether-cross-linked streptavidin binding ligands were designed from the crystal structure of a complex between streptavidin and a high affinity cyclic disulfide-cross-linked peptide ligand, cyclo- Ac-(CHPQGPPC)-NH2, originally discovered by phage display. Determination of the affinities of two chemically synthesized thioether ligands by surface plasmon resonance indicated affinities similar to the disulfide-cross-linked ligand from which they were designed. The crystal structures of the streptavidin—cyclic thioether complexes show that the nonlinker segments are bound in the same conformation as in cyc/o-Ac-(CHPQGPPC)-NH2 and make the same binding interactions with streptavidin as the disulfide-linked cyclic peptide. The structures, conformations, and dihedral energetic constraints of the thioether- and disulfide-cross-linked ligands are described and compared. Advantages of thioether cross-links over disulfide cross-links are discussed.

Permanent Setting of Washable Wool1

Samples of 100% wool fabric dimensionally stahilized by treatment with reactive polymers can be permanently set in the presence of alkaline catalysts and steam by procedure believed to induce cleavage of disulfide bonds and concurrent formation of hydrolytically stable thioether crosslinks by reaction with appropriate crosslinking agents. Polyfunctional reagents which are stable in aqueous alkaline solution, but react at elevated temperature with sulfhydryl groups, are suitable for this approach. They can be used in conjunction with alkaline catalyst to "sensitize" the stabilized fabric. Creases set in this manner are retained in home laundering.