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Biophysical Characterization of NEMO and IKK₂

  • Author(s): Drew, Devin Lee
  • et al.
Abstract

This dissertation describes the I-[kappa]B Kinase 2 (IKK₂) and NF-[kappa]B Essential Modulator (NEMO) proteins. Biophysical characteristics of N-terminal fragments of NEMO are described using methods that include circular dichroism (CD), equilibrium ultracentrifugation, and analytical gel filtration. Expression and purification of the N-terminal NEMO fragments are demonstrated along with the purification of exogenous IKK2 from an insect cell system. Purification of an active and stable fragment of IKK₂ lacking 89 C-terminal amino acids is also presented. Pulldown analysis and Isothermal Titration Calorimetry (ITC) experiments are implemented to demonstrate that NEMO 40-130 is capable of binding to the C-terminal 90 amino acids of IKK₂, while smaller fragments including NEMO 40- 90 and NEMO 60-120 are not. ITC data indicate that the association constant for NEMO 40-130 with GST-IKK₂ 665-756 is 4 x 10⁷ +/- 1 x 10⁷ M. Further details are found within the text of this document. Analysis of NEMO fragments by CD revealed characteristic double minima at 208 nm and 222 nm. These results form the first experimental confirmation of the software-predicted alpha-helical nature of the tested portions of NEMO. Thermal melts of NEMO 1-210 and 40-210 reveal a transition at roughly 40 ⁰C. This confirms the qualitative observation that the N-terminal fragments of E. coli expressed NEMO are unstable in a purified state. Thermal stability of NEMO 1-130 was too poor to be accurately measured by CD. Equilibrium analytical ultracentrifugation experiments indicate that NEMO 1-130 may exist as a tetrameric in equilibrium with monomers. Similarly, NEMO 1-210 appears form hexamers under the conditions tested. These data corroborate analytical gel filtration observations shared here that demonstrate a predominance of the tetrameric and hexameric oligomers for NEMO 1-130 and NEMO 1-210 respectively. This work presents the first in vitro observations of N-terminal fragments on NEMO and their interaction with the IKK₂ subunit. This work provides a foundation for further studies of IKK- complex structure and function

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