Spectroscopic characterization of membrane -docking C2 domains

• Main Author: Malmberg, Nathan John
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2004
• Subjects: Biochemistry; Biophysics
• ISBN: 0496143808; 9780496143801

C2 domains represent one of the most common Ca2+-signaling motifs in eukaryotic genomes. These domains typically bind multiple Ca 2+ ions in a small cleft formed by three Ca2+-binding loops, with the ions coordinated by oxygen atoms from aspartate and asparagines side chains. The Ca2+-bound domains exhibit a marked increase in membrane affinity, and are targeted to different cell membranes in their activated state. Proteins that transiently associate with membrane surfaces have been difficult to study by traditional structural characterization, but are accessible to study by EPR spectroscopy. To study the C2 domain of cytosolic phospholipase A2 by EPR, single cysteines were introduced at each site in the three Ca2+-binding loops and labeled with a thiol-specific nitroxide spin label. EPR spectra of the Ca2+-loaded and apo domains indicate that the differences between the two states are small and localized, consistent with an electrostatic mechanism of activation. EPR depth parameters measured for the membrane-docked C2 domain define the depth of insertion of each spin label into the membrane bilayer. The depth parameters illustrate the presence of α-helical structure in the membrane-bound protein. A model for the membrane-bound domain was calculated from depth parameters measured for membrane-inserting spin labels using a well-defined relationship between depth parameter and membrane depth. Ca2+ affinities for C2 domains with coordinating residues converted to cysteine were measured by monitoring the intrinsic tryptophan fluorescence of the domain while titrating the apo domain with Ca2+. The results, combined with theoretical calculations of the pKas of the coordinating aspartates, provide information about the order of Ca2+ binding, and the nature of cooperative binding of multiple Ca2+ ions in a small volume. Finally, spin labels were introduced into sites located throughout the three Ca2+ -binding loops of the C2 domain of protein kinase C-alpha. Depth parameters measured for these spin labels were used to generate a model for the membrane-docked C2 domain using a novel approximation of the depth parameter/membrane depth function. The model illustrates many of the similarities and differences in membrane interactions for different C2 domains.