Interactions Between Domains in Dematin, a Key Component of the Red Blood Cell Junctional Complex.
EMSL Project ID
25686
Abstract
The regulation and maintenance of the actin cytoskeleton is essential for all eukaryotic cells. The "villin-type headpiece" family of actin-associated proteins contains a modular C-terminal "headpiece" at their C-termini. The larger "core" domains of this family vary in sequence, size and function. This proposal will examine the binding and dynamic changes associated with the headpiece and core domains of dematin (band 4.9). Dematin is found in the red blood cell membrane (and other cells) and plays an important role in red blood cell stability. We have expressed and purified full-length dematin in both the 42 kDa (381 residues) and 52 kDa (403 residues) apparent molecular weight forms. Much of the dematin core domain is natively unfolded except for the headpiece domain and approximately 100 residues in the core domain that are not visible in 15N-HSQC spectra. The headpiece domain appears to be tethered to the core domain via a flexible linker sequence as there are no significant chemical shift changes in its resonances compared with the isolated domain. Our hypothesis is that those residues from the core missing in our spectra correspond to the trimerization domain. As the trimerization domain, those residues would have a high effective molecular weight resulting in broadened and weakened NMR signals due primarily to T2 (spin-spin) relaxation. We have also taken 15N HSQC spectra of a mutant (S381E) of dematin that mimics the phosphorylation of dematin headpiece that results in loss of actin bundling activity. Interestingly, in the mutant the resonances from the headpiece domain are not observable. This suggests that the headpiece domain has bound to the trimerization domain and its now much greater effective molecular weight results in the broadening of its resonances along with the trimerization domain. We are requesting NMR time at 800 MHz to run 15N-TROSY spectra to assist in localizing this trimerization domain and monitoring changes in resonances in the S381 mutant spectra. Additional time at 800 and 600 MHz is being requested to assign and determine the structure of the isolated trimerization domain. The proposal is in accord with the 'Biological Interactions & Dynamics' science theme as it will investigate the binding and large changes in dynamics associated with the binding of the small headpiece domain to the much larger trimerization domain found in the core.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-06-13
End Date
2008-07-11
Status
Closed
Released Data Link
Team
Principal Investigator