Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal structures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.

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The electrical potential due to fixed charge distributions is strongly altered in the vicinity of a membrane and notably dependent on aqueous electrolyte concentration. We present an efficient way to solve the nonlinear Poisson-Boltzmann equation applicable to general cylindrically symmetric dielectric geometries. It generalizes Gouy-Chapman theory to systems containing transmembrane channels. The method is applied to three channel systems: gramicidin, gap junction, and porin. We find that for a long, narrow channel such as gramicidin concentration variation has little influence on the electrical image barrier to ion permeation. However, electrolyte shielding reduces the image induced contribution to the energy required for multiple occupancy. In addition, the presence of electrolyte significantly affects the voltage profile due to an applied potential, substantially compressing the electric field to the immediate vicinity of the pore itself. In the large diameter channels, where bulk electrolyte may be assumed to enter the pore, the electrolyte greatly reduces the image barrier to ion permeation. At physiological ionic strengths this barrier is negligible and the channel may be readily multiply occupied. At all ionic strengths considered (l greater than 0.005 M) the image barrier saturates rapidly and is essentially constant more than one channel radius from the entrance to the pore. At lower ionic strengths (l less than 0.016 M) there are noticeable (greater than 20 mV) energy penalties associated with multiple occupancy.

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composition x,,, = 0.1 123 are put into eq 15, and the temperature T satisfying eq 15 is calculated by an iterative procedure. This temperature is the temperature of phase separation of the HA/D,O mixture under consideration. The calculation is repeated for mixtures of different compositions (different values of xoHA and Y) using the same value of xaB. The slight change of xap with Y is neglected. Figure 6 shows the result of these calculations.The calculated curve is shifted by 0.135 K to higher temperatures to overlap with the experimental data. The experimental data are the same as that shown in Figure 1. The same calculation is repeated for the system DA/H20 with x,,, = 0.1 123. The result is shown in Figure 7. Here, the calculated curve is shifted to higher temperatures by 0.205 K. The experimental data are the same as in Figure 2. The agreement between the form of the calculated and experimentally determined curves in Figures 6 and 7 is satisfactory. The temperature shifts of the calculated curves are assumed to reflect the influence of impurities.The properties of the coexisting surface of the system isobutyric acid/H20, D 2 0 appear to be typical for three-component systems in which H / D isotope-exchange reactions take place. This can be concluded from measurements of the temperature of phase separation as function of composition in the system phenol (OH)/D20 with an upper critical point" and the system 2-but-oxyethanol (OH)/D20 with a lower critical point.'* In both systems the mixture of critical composition has a lower D 2 0 content than the mixture of maximal and minimal phase-separation temperatures, respectively.2' For phenol (OH)/D20, T, = 78.445 "C, XOHP,~ = 0.095, T,,,,, = 78.495 "C, and x~H~,~~~ = 0.085. For 2-butoxyethanol (OH)/D20, T, = 42.385 "C, xoHBp = 0.061, and Tp,min = 42.350 OC.Numerical solutions to the hypernetted chain (HNC) integral equation have been obtained for a model system representing an infinitely dilute rodlike polyelectrolyte in an aqueous 1-1 electrolyte solution. Bulk salt concentrations, cST, of lo-], and M, and reduced polyion charges, 6, ranging from 0.5 to 5.0, have been studied. The distribution functions are analyzed in terms of various structural parameters and compared with the counterion condensation (CC) formalism of Manning and with available Poisson-Boltzman (PB) data. Although HNC and CC are in general qualitative agreement, several quantitative aspects of CC theory, including the special nature off = 1.0 and the csT independence of the Manning radius, are not reproduced by HNC.