Enzymes R Us: BIOMOD at Rutgers University

1 Project Overview

Our project had two phases: a computational phase in which modifications to protein structure were modeled and the most energetically favorable protein structures were selected. And, a wet lab phase in which the gene of the proposed computational designs were constructed, expressed, and their protein products were characterized using analytical methods.

In order to promote the formation to large macromolecular assemblies, binding domains capable of forming covalent bonds were developed. Binding domains are protein domains found in cellular signaling pathways that bind non-covalently to peptides. Non-covalent binding limits the formation of large assemblies, since the peptide exists in equilibrium between bound and non-bound states. Covalent binding enables the formation of large assemblies, since once bound, the peptide does not become unbound from the domain even in reducing environments.

After we developed these designed binding domains, they were inserted into the loop regions of fluorescent proteins (FPs). At either the amino [N] or carboxyl [C] terminus of the FP, the peptide of the FP was appended with a triple glycine spacer. We then used structural alignment and clash check algorithms to model potential assemblies of FPs that may form with various designed binding domains.

2 Methods and Implementation

Below are methods for the dry lab and the wet lab. Dry lab protocols contain the relevant Rosetta Scripts xml files and Python scripts used to generate models. Wet lab protocols and strategy detail the steps taken to express the biological constructs.

2.1 Dry Lab

2.2 Wet Lab

3 Results and Discussion