| Summary: | The focus of this project is to develop a greater understanding of the protein-protein interaction between the measles Nucleocapsid Binding Domain (NBD) protein and its ligand N-protein, which are part of the paramyxovirus replication complex. This interaction is an example of a binding reaction where one partner (the N protein) is intrinsically unstructured, but undergoes a coil-to helix transition upon binding. The NBD’s biological role is to bind the N protein which coats the viral RNA genome. However, it must quickly release as the replication complex moves along the RNA during replication. To facilitate this biological function, NBD displays a weak and short-lived interaction with the N-protein that is heavily dependent on the structural stability of both proteins. We are utilizing this pair of proteins as a model system for the study of coupled folding and binding processes. In this work, we have investigated the influence of helix formation in N protein on the NBD-N interaction. Helix stabilizing and destabilizing mutations were introduced into the NBD binding site on the measles N-protein in order to analyze the effects that the structural stability of N-protein have in the binding equilibrium between NBD and N. The measles N protein was mutated by site directed mutagenesis into an unstable mutant, L496G, and a more favorably stable mutant, L496A. These were fused to a small protein, SUMO, to facilitate purification. After, induction and expression of the desired proteins, the proteins were extracted from their respective transformed E. coli BL21-D3 gold expression strain and submitted to protein purification techniques such as Co²⁺ affinity column chromatography, dialysis, and centrifuge concentration. The purity and success of the purification was evaluated by SDS-PAGE electrophoresis, and absorption spectroscopy. The binding reaction between measles NBD wild type, N protein and the mutated variants was analyzed by isothermal titration calorimetry (ITC).
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