Biophysical characterization of NEMO and IKK2.

摘要

This dissertation describes the IkappaB Kinase 2 (IKK2) and NF-kappaB 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°C. 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 tetramer 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.