The relationship between latent secondary structure within an unbound intrinsically disordered protein and the thermodynamics of complexation with a target molecule
Open Access
Author:
Zidell, Anthony Wallace
Graduate Program:
Chemistry
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
March 16, 2015
Committee Members:
Scott A Showalter, Thesis Advisor/Co-Advisor
Keywords:
intrinsically disordered protein thermodynamics isothermal titration calorimetry circular dichroism spectroscopy
Abstract:
Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their target molecules. Despite their plasticity, disordered proteins have often been observed in the unbound state to possess latent structure resembling the folded bound state. However, the role of this preformed structure and its effect on the thermodynamics of binding are currently not well understood. Our group has recently shown through temperature-series isothermal titration calorimetry that the folding-upon-binding of a model disordered protein, FCP1, to its target, RAP74, is equivalent to the cooperative folding of a globular protein. Changes in molar enthalpy upon formation of the FCP1–RAP74 complex were deconstructed into contributions from the primary driving force of that interaction, the hydrophobic effect, and other concomitant driving forces, such as those associated with helix formation.
In the current work, we attempt to parse the contribution of helix formation to stabilization of the IDP–target complex through the generation of a helicity series by rational mutagenesis of the disordered protein. In this way, a panel of FCP1 mutants is prepared with amino acid substitutions that confer modulated helicity without disrupting the hydrophobic binding face. The extent of latent helical structure within each FCP1 variant has been quantified using circular dichroism spectroscopy and shown to be linearly correlated with various thermodynamic parameters derived from isothermal titration calorimetry and urea denaturation of the FCP1–RAP74 complex. These observations allow us to better understand the contribution of latent secondary structure in an IDP to the energetics of IDP–target complex formation.
