Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site.
The zinc-specific fluorophore, Zinpyr-1, is used in competition assays to determine the kinetic and thermodynamic parameters of Zn2+ binding to engineered bi-Histidine sites located in Ubiquitin and the B domain of protein A (BdpA). These binding sites are used in ψ-analysis studies to investigate structure formation in the folding transition state identified by the change in folding rate upon the addition of metal ions. For Ubiquitin, the on-rate binding constant and binding affinity for a site located along an α-helix are measured to be ~107 M-1s-1 and 3 μM, respectively. For a site located across two β-strands, the metal binding affinity was too weak to measure in the dye competition assays (Kd > 55 μM). The equilibrium-determined values for the Zn2+-induced stabilization of Ubiquitin and BdpA match the values derived from changes in the global folding and unfolding rates. Therefore, metal-ion binding is in fast equilibrium during the transit over the free energy barrier. Accordingly, the folding rate must be slower than the product of the fractional population of a high energy intermediate with the metal site formed and the metal binding on-rate constant. The known folding rate of 20 s-1 at 1.5 M guanidinium chloride in 400 μM Zn2+ provides an upper bound for the stability of such intermediates, ΔGU-I < +4 kcal·mol-1. These results support a view of the apparent two-state protein folding reaction surface as a fast pre-equilibrium between the denatured state and a series of high energy species. The net folding rate is a product of the equilibrium constant of the highest energy species and a transmission rate. For Ubiquitin, we estimate the transmission rate to be ~104 s-1. Implications to the role of unfolded chain diffusion on folding rates and barrier heights are discussed.
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