NMR investigation of folding and dynamics of the IkB/NFkB system
EMSL Project ID
3152
Abstract
More than 36 % of eukaryotic proteins are expected to be natively unfolded or only partially folded in their functional free state but well-structured when bound to a cellular target. Binding-coupled folding has therefore been recognized as an essential mechanism to control function. The NFKB/IKB system represents a prototype for this large class of proteins, as demonstrated by preliminary MS, CD and NMR data strongly suggesting that the free state of IKBa is only partially folded. This is in marked contrast with the crystal structure of the IKBa-p65/p50 NFKB complex, in which IKBa adopts an ankyrin repeat conformation. Considering the medical relevance of the NFKB/IKB system for immune response, inflammation, cellular growth, differentiation, cell adhesion and apoptosis, we now plan to map the structural and dynamical variations that occur as IKBa and NFKB advance in the complex energy landscape that connects their free states to their bound forms. In view of the intrinsic flexibility of these states we anticipate that NMR will be suitable for this investigation and specifically our aims are: (1) To assign the 1H, 15N and 13C nuclei of the functional free state of IKBa using triple-resonance NMR methods; (2) To characterize the backbone dynamics of free IKBa using 15N relaxation NMR measurements analyzed according to the reduced spectral densities approach; (3) To map the structural propensities of free non-globular IKBa. Short-range interactions will be characterized using mainly secondary chemical shifts, NOEs and H/H or H/D exchange with water, while long-range residual order will be probed by residual dipolar couplings, mutations and paramagnetic relaxation enhancements; (4) To characterize by NMR both the free and the equilibrium partially folded non-native bound states of the NFKB sequences involved in nuclear localization, which are known to adopt different conformations when bound to different targets ("contingent folding"). We plan to extend the analyses in aims (1-4) to different mutants and isoforms with altered conformational preferences. In addition, we plan to integrate the NMR results with complementary MS, CD and fluorescence anisotropy measurements (cfr. Komives aims 1-3 and Ghosh aim 3) and to interpret all the experimental data on the basis of the ensemble of structures provided by the simulations carried out by the Wolynes group (cfr. Wolynes aims 1- 4). The ensuing structural and dynamical map will then be correlated with the binding thermodynamics, kinetics and proteolytic stability of the NFKB/IKB complexes (cfr. Ghosh aim 1 and Hoffman aims 1-2). It is anticipated that this investigation will further the understanding not only of the NFKB/IKB system, but also of non-globular intrinsically unstructured proteins in general.
Project Details
Project type
Capability Research
Start Date
2002-12-16
End Date
2004-03-09
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members