A novel structure-based approach to study the structure and dynamics of flexible multi-domain monomeric protein kinases, which otherwise do not yield diffraction quality crystals, is described. A combination of segmental 15N-isotopic labeling of a regulatory domain with site-directed paramagnetic nitroxide spin labeling of the kinase domain is employed. Nuclear magnetic resonance studies of the enhancement of amide proton relaxation rates of the 15N-isotopically labeled regulatory domain caused by insertion of the paramagnetic nitroxide spin label on the kinase domain provide long-range distance restraints for determination of both the average positional structure and the relative flexibility exhibited between the two contiguous domains. Clefts and crevices detected around the dynamic domain-domain interface provide new targeting sites for tethered-based extension of current small-molecule lead compounds to produce more potent and selective pharmaceutical agents.
|Original language||English (US)|
|Number of pages||27|
|Journal||Methods in molecular biology (Clifton, N.J.)|
|State||Published - 2006|
ASJC Scopus subject areas
- Molecular Biology