Preparation and crystallization of dynamic NF-kappa B center dot I kappa Bcomplexes

Citation
T. Huxford et al., Preparation and crystallization of dynamic NF-kappa B center dot I kappa Bcomplexes, J BIOL CHEM, 275(42), 2000, pp. 32800-32806
Citations number
36
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
275
Issue
42
Year of publication
2000
Pages
32800 - 32806
Database
ISI
SICI code
0021-9258(20001020)275:42<32800:PACODN>2.0.ZU;2-H
Abstract
The formation of single, well-diffracting crystals is a requirement for any molecular structure determination by x-ray crystallography. Crystallizatio n of biological macromolecules can represent a significant obstacle when th e subject exhibits internal flexibility or indiscriminate self-association. In such cases, the removal of inherently flexible regions and the addition of stabilizing ligands can improve the probability of crystal formation an d ordered growth. We have applied these principles in order to form crystal s of the Rel homology region of transcription factor NF-kappaB in complex w ith its inhibitors I kappaB alpha and I kappaB beta. None of these molecule s crystallizes in the absence of a binding partner. Recombinant overexpress ion of truncated I kappaB alpha required selection of the correct start sit e. NF-kappaB .I kappaB alpha complex crystals formed under relatively strin gent conditions. NF-kappaB .I kappaB beta complex crystals were formed by a nalogy to NF-kappaB .I kappaB alpha, although some modifications in purific ation and complex formation were necessary due to differences between the i nhibitors.