An optimized ATP/PP(i)-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis.

We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains.

Complex with a phage display-derived peptide provides insight into the function of insulin-like growth factor I.

The dramatic improvement in the NMR spectra of insulin-like growth factor I (IGF-I) in the presence of a peptide identified from a phage display library has allowed for the first time the determination of a high-resolution solution structure for much of IGF-I. The three helices of IGF-I in this complex have an arrangement similar to that seen in high-resolution crystal structures of IGF-I and insulin, although there are differences in the conformation and precise location of helix 3. A cluster of hydrophobic and basic side chains within the turn-helix motif of the peptide contact a hydrophobic patch on helices 1 and 3 of IGF-I.

Rapid identification of small binding motifs with high-throughput phage display: discovery of peptidic antagonists of IGF-1 function.

A panel of 22 naïve peptide libraries was constructed in a polyvalent phage display format and sorted against insulin-like growth factor-1 (IGF-1). The libraries were pooled to achieve a total diversity of 4.4 x 10(11).