Biomedical subjects
Publications and source records attributed to Barbara Imperiali.
6-(2-Dimethylaminonaphthoyl) alanine (DANA) was prepared via an enantioselective synthesis and incorporated into the S-peptide of RNase S establishing the large changes in fluorescence that can occur upon peptide-protein interaction.
[reaction: see text] An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.
The role of a single glycine hinge residue in the structure of BBAT1, a beta(beta)(alpha) peptide that forms a discrete homotrimeric structure in solution, was evaluated with 11 new peptide sequences which differ only in the identity of the residue at the hinge position. The integrity of the structure and oligomeric state of the peptides was evaluated by using a combination of analytical ultracentrifugation and circular dichroism spectroscopy. Initially, it was discovered that the glycine hinge adopts backbone dihedral angles favored in D-amino acids and that incorporation of D-alanine at the hinge position stabilizes the trimer species. Subsequently, the effect of the side chains of different D-amino acids at the hinge position was evaluated. While incorporation of polar amino acids led to a destabilization of the oligomeric form of the peptide, only peptides including D-Ser or D-Asp at the hinge position were able to achieve a discrete trimer species. Incorporation of hydrophobic amino acids D-Leu and D-Phe led to oligomerization beyond a trimer to a tetrameric form. The dramatic differences among the thermodynamic stabilities and oligomeric states of these peptides illustrates the pivotal role of the hinge residue in the oligomerization of the beta(beta)(alpha) peptides.
Linear hexapeptides featuring the asparagine mimetics alanine-beta-hydrazide, alanine-beta-hydroxylamine, and 1,3-diaminobutanoic acid have been synthesized as oligosaccharyl transferase (OT) substrate mimetics and chemoselectively N-glycosylated to obtain the corresponding neoglycopeptides as OT product mimetics. The effect of glycosylation on the binding of these asparagine surrogates is in stark contrast with the effect of modification of native asparagine. In native N-linked glycosylation, product inhibition is minimal and glycopeptides show very low affinity for OT. In contrast, glycosylation of the substrate mimetics maintains or even improves affinity of the corresponding product mimetic for OT. Conformational considerations suggest that the flexibility of the N-glycosyl linkage in these neoglycopeptides allows them to be accommodated in the OT binding site while the native trans glycosyl amide linkage is rejected. These results provide insight into how OT minimizes product inhibition, thereby ensuring effective substrate turnover.
Oligosaccharyl transferase is part of the macromolecular machinery that processes nascent proteins in the endoplasmic reticulum. The enzyme is highly conserved, catalyzes the initial step in the biosynthesis of N-linked glycoproteins and acts as a 'gatekeeper' for the secretory pathway. As more proteins associated with oligosaccharyl transferase are identified, the intricacies of the enzyme and the relationship with other proteins in the lumen of the endoplasmic reticulum are starting to be unraveled.
The syntheses of two new nonstandard amino acids, Flu (6) and XBp (20), and a new synthesis of Dmd (12) are reported. These residues exhibit fluorescence, metal-coordination, and fluorescence-quenching properties, respectively. These building blocks have been incorporated into peptides via solid phase peptide synthesis to afford the prototype for a photoinduced electron transfer-based metal ion chemosensor. The fluorescence of the peptides is modulated upon metal binding. This results from a metal ion-induced conformational change that brings the side chains of the Flu and Dmd amino acids into proximity, thereby favoring photoinduced electron transfer (PET) fluorescence quenching.