General workflow for students investigating the noncovalent interactions involved in P450_sky-catalyzed β-hydroxylation of L-(OMe)-Tyr.

<p>This involves computational analysis (Step 1), molecular biology or synthetic chemistry (Step 2), protein purification (Step 3), chemoenzymatic assays (Step 4), and biochemical and biophysical experiments (Step 5). This workflow is a template for realizing an integrated science curriculum, as described and assessed by the Interdisciplinary Learning Consortium [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003145#pbio.2003145.ref009" target="_blank">9</a>]. PCP, peptidyl carrier protein.</p

Structures of the skyllamycin NRPS PCP domain (PCP7_sky, green) bound to a hydroxylating cytochrome P450 (P450_sky, multicolored).

<p>Students visualized this structure in PyMOL (A) and evaluated the roles of 4 amino acid residues at the P450_sky–PCP7_sky interface (B). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003145#pbio.2003145.box002" target="_blank">Box 2</a> for details. NRPS, non-ribosomal peptide synthetase; PCP, peptidyl carrier protein.</p

In a semester of Biochemistry Superlab, students investigated the protein–protein interactions involved in the β-hydroxylation of the natural product skyllamycin.

<p>The skyllamycin peptide is constructed by <i>Streptomyces</i> bacteria via a NRPS involving 11 biosynthetic modules (“M”), composed of catalytic domains such as the A, PCP, and C domains. The <i>in trans</i> cytochrome P450 (P450_sky, orange) interacts with PCP-bound amino acids on modules 5, 7, and 11 to install β-hydroxyl groups (highlighted in orange on the structure of skyllamycin, right). As a class, we tackled the central question: What is the biochemical basis for the selectivity of the interaction of PCP from module 7 with P450_sky to install the hydroxyl group on the L-(OMe)-Tyr (incorporated at the boxed position of skyllamycin)? A, adenylation; C, condensation; NRPS, non-ribosomal peptide synthetase; PCP, peptidyl carrier protein.</p

SV-AUC data collected and analyzed by students to obtain dissociation constants for P450_sky and mutants of P450_sky interacting with inhibitor-bound PCP7_sky (L-imidazoyl-PCP7_sky).

<p>Comparisons of the <i>c(s)</i> distributions are shown for 10 μM P450_sky wild type alone and in complex with 60 μM L-imidazoyl-PCP7_sky L62A, L-imidazoyl-PCP7_sky F66A, and L-imidazoyl-PCP7_sky wild type. A) 280 nm (protein), B) 418 nm (heme). In general, shifts to the right suggest that the reaction boundary favors tighter binding. SV-AUC, sedimentation velocity experiments with an analytical ultracentrifuge.</p