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A methodology to quantify the binding of proteins to target DNA molecules based on single-molecule detection and real-time counting of individual free and bound fluorescently tagged molecules flowing past a detection device is presented. The methodology was used for measuring DNA binding by fluorescently tagged domains of four distinct transcription factors and these proteins represent different structural classes, quaternary states and relative affinities.

Research has been conducted on the cytosolic phospholipase A (sub)2-alpha C2 domain. The new model for the penetration and orientation depth of the C2 domain at the membrane interface developed via the use of the site-directed spin labeling is described.

Research shows that the high calcium selectivity of the cytosolic phospholipase A(sub)2 is influenced by cation charge and size and takes two metal ions to form a complex between the enzyme and the membrane. Structural aspects of the metal ion-binding site of the C2 domain of the enzyme are discussed.

Association and dissociation kinetics of the membrane binding by the isolated C2 domain of protein kinase C beta and the full-length enzyme betaII reveal that calcium targets the enzyme via the C2 domain, which triggers C1 domain to search for the secon messenger diacylglycerol.

Research delineates the functional aspects of the specialized calcium-triggered membrane-docking modules, C2 domains of the signaling proteins. Data indicate that the domains of the three signaling proteins differ in their calcium affinity, kinetics, membrane affinity, and head group selectivity suggesting that the domains adapt to different contexts of distinct cellular signals.

The location of the membrane-docking face on the Ca2+-activated C2 domain of cytosolic phospholipase A was investigated. The experiment utilized 16 engineered cysteines that were individually substituted for solvent-exposed residues spanning all surface regions of the cPLA2 C2 domain. Results showed that docking of C2 domains is stimulated by the binding of multiple Ca2+ ions to a conserved set of residues found within three otherwise variable Ca2+-binding loops.

The role of cytosolic phospholipase A2 (cPLA2 C2) domain in the Ca2+ ion binding was determined through utilization equilibrium binding and stopped-flow kinetic experiments. It was shown that Ca2+-regulated C2 domains typically bind two Ca2+ ions with positive cooperativity resulting in conformational change and membrane docking. Intrinsic fluorescence of Trp (super 71) was enhanced following binding of Ca2+ to the domain in the

Studies cation charge and size selectivity of the C2 domain of cytosolic phospholipase A(sub 2). Role of cation charge and size constraints in high calcium(sup 2+) selectivity of the C2 domain; Role of two bound multivalent metal ions in the formation of a cPLA(sub 2)-alpha C2 domain; Impact of the unique structural features of the metal ion-binding site in the C2 domain.

Compares the equilibrium and kinetic parameters of C2 domains isolated from three functionally distinct signaling proteins. Functional and mechanistic diversity of C2 domains from different calcium signalling pathways; Regulation of diverse processes critical for cell signaling; Calcium coordination and contrasting mechanisms of membrane docking.