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Specificity and temporal control of transcriptional machinery are encoded within sequence-specific transcription factors, of which there are thousands in mammalian genomes. Efforts to completely decipher this code will require an understanding of the DNA binding thermodynamic and kinetic properties displayed by each transcription factor, a daunting task given the current methodologies for measuring these interactions. Here, we present a novel 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. Using this technology, we measured DNA binding by fluorescently tagged domains of four distinct transcription factors, namely, human early growth response protein Egr-1, vertebrate GATA-1, Drosophila GAGA factor, and λ bacteriophage Cro repressor. These proteins represent different structural classes (zinc-finger and helix-turn-helix), quaternary states (monomeric and dimeric), and relative affinities (high, intermediate, and low). Specific binding of each protein to its cognate DNA target was demonstrated at low picomolar concentrations. The equilibrium (Kd) and kinetic (kon and koff) constants governing DNA binding by one of these transcription factors, that of Egr-1, were measured using this approach. Kd values obtained from three different types of saturation titrations were reproducible and consistent, yielding values between 10 and 14 pM that, along with the kinetic constants, agree closely with literature values. Because this methodology offers several significant advantages over other existing approaches, namely, real-time determination, requirement for small amounts of reagents, high reproducibility, exquisite sensitivity, and amenability to high-throughput analysis, it is suitable for characterizing DNA-binding proteins as well as other interacting pairs of molecules that can be fluorescently tagged. [ABSTRACT FROM AUTHOR]

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.

Investigates the mechanism of the Ca[sup2+]- induced binding of the PKC beta II to the anionic membranes of C2 domain. Role of the electrostatic interaction on the higher-affinity membrane-bound complex; Revelations of the Ca[sup2+]n induced binding full-length protein; Molecular basis for the cofactor regulation of the PKC.

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.

Provides information on a study which determines the outline of the membrane-docking face of the C2 domain module present in eukaryotic cells. Materials and methods of the study; Summary of results; Discussion.

Examines the Ca2+-signaling cycle of a membrane-docking C2 domain. Eukaryotic signal-transducing proteins; Vertebrate inflammation pathway; Hydrolyzation of specific lipids to release arachidonic acid; Initiation of inflammatory response.

Abstract: Single molecule detection of target molecules specifically bound by paired fluorescently labeled probes has shown great potential for sensitive quantitation of biomolecules. To date, no reports have rigorously evaluated the analytical capabilities of a single molecule detection platform employing this dual-probe approach or the performance of its data analysis methodology. In this paper, we describe a rapid, automated, and sensitive multicolor single molecule detection apparatus and a novel extension of coincident event counting based on detection of fluorescent probes. The approach estimates the number of dual-labeled molecules of interest from the total number of coincident fluorescent events observed by correcting for unbound probes that randomly pass through the interrogation zone simultaneously. Event counting was evaluated on three combinations of distinct fluorescence channels and was demonstrated to outperform conventional spatial cross-correlation in generating a wider linear dynamic response to target molecules. Furthermore, this approach succeeded in detecting subpicomolar concentrations of a model RNA target to which fluorescently labeled oligonucleotide probes were hybridized in a complex background of RNA. These results illustrate that the fluorescent event counting approach described represents a general tool for rapid sensitive quantitative analysis of any sample analyte, including nucleic acids and proteins, for which pairs of specific probes can be developed. [Copyright &y& Elsevier]

Focuses on the C2 domain of key signaling proteins during intracellular calcium signals. Membrane orientation of C2 domain by site-directed spin labeling; Determination membrane depth parameters; Generation of a model for the position of the C2 domain at the membrane interface.