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Biophysical characterization of NEMO...

Drew, Devin Lee.

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Biophysical characterization of NEMO and IKK2.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Biophysical characterization of NEMO and IKK2./
Author:	Drew, Devin Lee.
Description:	140 p.
Notes:	Adviser: Gourisankar Ghosh.
Contained By:	Dissertation Abstracts International67-10B.
Subject:	Biophysics, General. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3239883
ISBN:	9780542944307

Biophysical characterization of NEMO and IKK2.
Drew, Devin Lee.

Biophysical characterization of NEMO and IKK2. - 140 p.

Adviser: Gourisankar Ghosh.

Thesis (Ph.D.)--University of California, San Diego, 2007.

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.

ISBN: 9780542944307Subjects--Topical Terms:

1019105
Biophysics, General.

Biophysical characterization of NEMO and IKK2.
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005 20110920
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020 $a 9780542944307
035 $a (UMI)AAI3239883
035 $a AAI3239883
040 $a UMI $c UMI
100 1 $a Drew, Devin Lee. $3 1293434
245 1 0 $a Biophysical characterization of NEMO and IKK2.
300 $a 140 p.
500 $a Adviser: Gourisankar Ghosh.
500 $a Source: Dissertation Abstracts International, Volume: 67-10, Section: B, page: 5725.
502 $a Thesis (Ph.D.)--University of California, San Diego, 2007.
520 $a 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.
520 $a 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.
520 $a 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.
520 $a 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.
520 $a 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.
590 $a School code: 0033.
650 4 $a Biophysics, General. $3 1019105
650 4 $a Chemistry, Biochemistry. $3 1017722
650 4 $a Health Sciences, Immunology. $3 1017716
690 $a 0487
690 $a 0786
690 $a 0982
710 2 0 $a University of California, San Diego. $3 1018093
773 0 $t Dissertation Abstracts International $g 67-10B.
790 $a 0033
790 1 0 $a Ghosh, Gourisankar, $e advisor
791 $a Ph.D.
792 $a 2007
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3239883