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Figure 11.17. The predicted macroscopic current. The rate constants that have been fitted to results from equilibrium recordings (see Figs. 11.14—11.16) were used to calculate the macroscopic response to a 0.2-ms pulse of ACh (1 toM), as in Colquhoun and Hawkes (44). This calculation predicts that the mutation will cause a sevenfold slowing of the decay of the synaptic current, much as observed (102).

Figure 15.40. CPK structures illustrating the sequential metabolism of the molecular package used for brain delivery cf a leucine enkephalin analog.

Figure 15.11. Overlapping pharmacophore structures of corticosteroids, (a) Clobetasol propionate (in gray) and the soft corticosteroid loteprednol etabonate (26) (in black). The view is from the a side, from slightly below the steroid ring system, (b) Loteprednol etabonate (in black) and a 17a-dichloroester soft steroid (in gray). This view is from the p side, from above the steroid ring system.

Figure 15.11. Overlapping pharmacophore structures of corticosteroids, (a) Clobetasol propionate (in gray) and the soft corticosteroid loteprednol etabonate (26) (in black). The view is from the a side, from slightly below the steroid ring system, (b) Loteprednol etabonate (in black) and a 17a-dichloroester soft steroid (in gray). This view is from the p side, from above the steroid ring system.

Figure 15.40. CPK structures illustrating the sequential metabolism of the molecular package used for brain delivery cf a leucine enkephalin analog.

Figure 16.7. Graphical user interface of ArrayScan HCS System. Staurosporine treatment of BHK cells in 96-well microplate. Drug concentrations increase from left to right (controls in first two columns). Well color coding reflects (1)well status (current well being scanned is shown in white whereas unscanned wells are shown in grey) and (2) well results (pink wells have insufficient cell number, blue wells are below user-defined threshold range, red wells are above range, and green wells are in range for the selected cell parameter). Images correspond to current well, with three monochrome images reflecting three respective fluorescence channels, and composite (three-channel) color overlay in upper left panel. Nuclei are identified by the Hoechst dye (blue), microtubules by fluorescently labeled phalloidin (green),and mitochondria by Mitotracker stain (red).

Figure 16.7. Graphical user interface of ArrayScan HCS System. Staurosporine treatment of BHK cells in 96-well microplate. Drug concentrations increase from left to right (controls in first two columns). Well color coding reflects (1)well status (current well being scanned is shown in white whereas unscanned wells are shown in grey) and (2) well results (pink wells have insufficient cell number, blue wells are below user-defined threshold range, red wells are above range, and green wells are in range for the selected cell parameter). Images correspond to current well, with three monochrome images reflecting three respective fluorescence channels, and composite (three-channel) color overlay in upper left panel. Nuclei are identified by the Hoechst dye (blue), microtubules by fluorescently labeled phalloidin (green),and mitochondria by Mitotracker stain (red).

m D94, C-terminus J32 to K-lOa:-^. Upon oxygen-K ation the Fe(II) atom moves into the plane of ■ the heme. The proximal His also reorients and I moves with the Fe(II) as does the F helix (of | which the His is a part). For the F helix to I move, a rearrangement at the a±fi2 interface is | necessary. The contact between the H97 of the | /3-chain to the T41 of the a-chain must disso-- ciate. H97 then is able to form a contact with T38, one turn down the helix. This movement j is much like the knuckles of two hands sliding ever one another. The result of these tertiary movements is a gross 15' rotation relative to the ajjSi dimer to the a2fi2 dimer and a narp rowing of the central solvent-filled cavity. f Physiologically, the cooperativity of 0, bind! ing allows Hb to take up and release 02 over a small range of 0, pressures, comparable to pressure changes between the lungs and target tissues. (For a complete review of hemoglo-!;■ bin, refer to Ref. 16 and references therein.)

The binding of 02 to hemoglobin can be mathematically explained by both the MWC l and KNF/DE models. Given the crystallo-graphic snapshots of the R and T states and I abundant biochemical data, which model best [ explains the cooperativity in Hb? Both.

The quaternary transition of Hb between the T and R state is consistent with the concerted or MWC model. The two potential interactions between His-97 of the j3-chain and either T41 or T38 of the a-chain sterically does not allow intermediary states within the tet-ramer. Coupled with the constraints of the i more rigid and a2)32 interfaces, this bi-1 nary switch (T41 or T38) forces the transition of the entire molecule concomitantly, T —» R.

The X-ray structure of human hemoglobin, in which only the a-subunits are oxygenated, offers some evidence for an induced-fit model (11). The partially oxygenated structure rei sembles the T state, with the exception of the ; a-chainFe(II) atoms, which have moved about 0.15 A closer to the heme plane. Perutz had ; postulated that the movement of the Fe(II) I atom into/out of the heme plane in the oxy/ states would exert a tension on the atom proximal His bond. This tension was measured spectroscopically by setting up Hb with NO, which binds with higher affinity than 0„ to pull Fe(II) into the plane of the heme and IHP, which acts as a mimic of BPG

and induces the T state, to pull Fe(II) out of the plane. The opposing forces on the Fe(II), if it triggers the transition from T —> R, would exceed the strength of the bonds and they would break. Therefore, the observation of the human Hb intermediary position of the Fe(II) atom suggests that as more 02 binds to the T state, this tension builds within the tertiary structure until the molecule is forced to undergo the T —> R transition.

In part Hb fits both the MWC and KNF/DE models and underlies the premise that the models provide a starting point for interpreting the mechanistic machinations of a protein, but cannot be exclusive of one another or other potential intermediates. The examination of Hb's transition between its "T" and "R" states exemplifies the means by which proteins may transmit and communicate within and between subunit's ligand binding events. To summarize, Hb utilizes salt bridge formationldisruption at the C terminus, movement of secondary structure (F helix) induced by ligand coordination to a metal, and changes in a subunit-subunit interface (a^) that, although not changing the nature of the hydrogen bonding pattern, does trigger gross quaternary movement. The knowledge of Hb's T R transition and other proteins, such as ATCase, form the basis of structural changes that can be used to identify triggering mechanisms in other allosterically regulated and cooperative processes in proteins.

Although the above discussion has focused primarily on ligand affinity, it should be pointed out that the induced conformational changes or the differences between preexistent states may be reflected in altered catalytic activity, not just ligand affinity. This latter point illustrates another aspect of allosteric regulation, which can best be described in the concepts of K-type and V-type effects. A K-type effect is one that affects ligand affinity, whereas a V-type effect is one that affects the catalytic activity of the protein. Although the majority of allosteric enzymes are K-type systems, in recent years a number of V-type enzymes have been identified, and in many ways may be the more important as potential targets for drug design.

[Bound ligand]/[Free ligand] a- rate/[Substrate]

Figure 9.6. Scatchard or Eadie-Hofstee plots of data indicative of no cooperativity, negative coop-erativity and positive cooperativity.

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