Characterization of the nucleotide binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase
Subtitle: probing effects of nucleotide by NMR.
Published: 2004
Language: eng
Number of pages: 156
Description: In muscle cells, the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) plays a key role in restoring cytoplasmic Ca2+ levels to the resting concentration after a transient surge caused by excitation-contraction coupling. The mechanism by which Ca2+ is translocated to the lumen of the ER involves major conformational rearrangements among the three cytoplasmic domains: actuator (A), nucleotide binding ( N), and phosphorylation (P) domains; and within the transmembrane Ca2+-binding domain of SERCA.Large scale-production of SERCA-N enabled us to study this functionally essential domain by NMR spectroscopy, in order to answer specific questions about local effects of ligand binding in solution. Essentially complete backbone  1HN, 13Calpha,  13Cbeta, and 15N resonance assignments have been obtained for SERCA-N by means of heteronuclear multidimensional NMR techniques. Chemical shift-derived secondary structure analysis of SERCA-N established that the overall 2&deg; structure in solution remained essentially the same when compared to that of the crystal structure. Distortion of two beta strands was noted at two sites: beta6 and beta12. NMR titration experiments with AMP-PNP identified at least 29 amino acids within a confined nucleotide-binding site. These results were in excellent concurrence with site-directed mutagenesis studies, and present more comprehensive evidence for the involvement of other amino acid residues within that region.15N backbone relaxation experiments on the free and nucleotide-bound SERCA-N identified site-specific changes in backbone dynamics that were seen to converge on the central beta sheet domain. Nucleotide binding produced diverse effects on dynamics; causing both enhanced mobility and rigidifying effects at various sites. Taken together with spectroscopic studies, these results indicate that the overall fold and backbone motional properties of SERCA-N remained essentially the same in the presence of AMP-PNP, and reveal evidence for internal counterbalancing effects on backbone dynamics upon binding the nucleotide, which propagate through the central beta sheet.
Notes: Adviser:  David H. MacLennan.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-10, Section: B, page: 5033.