Uncovering protein-protein interactions through a team-based undergraduate biochemistry course

Abstract

How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.

Fig 1. In a semester of Biochemistry Superlab, students investigated the protein–protein interactions involved in the β-hydroxylation of the natural product skyllamycin.

The skyllamycin peptide is constructed by Streptomyces bacteria via a NRPS involving 11 biosynthetic modules (“M”), composed of catalytic domains such as the A, PCP, and C domains. The in trans 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.

Fig 2. Structures of the skyllamycin NRPS PCP domain (PCP7_sky, green) bound to a hydroxylating cytochrome P450 (P450_sky, multicolored).

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 Box 2 for details. NRPS, non-ribosomal peptide synthetase; PCP, peptidyl carrier protein.

Fig 3. General workflow for students investigating the noncovalent interactions involved in P450_sky-catalyzed β-hydroxylation of L-(OMe)-Tyr.

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 [9]. PCP, peptidyl carrier protein.

Fig 4. 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).

Comparisons of the c(s) 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.