Different dark conformations function in color-sensitive photosignaling by the sensory rhodopsin I-HtrI complex.

The haloarchaeal phototaxis receptor sensory rhodopsin I (SRI) in complex with its transducer HtrI delivers an attractant signal from excitation with an orange photon and a repellent signal from a second near-UV photon excitation. Using a proteoliposome system with purified SRI in complex with its transducer HtrI, we identified by site-directed fluorescence labeling a site (Ser(155)) on SRI that is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser(155)Cys with a nitroxide side chain, we detected a change in conformation following one-photon excitation such that the spin probe exhibits a splitting of the outer hyperfine extrema (2A'(zz)) significantly smaller than that of the electron paramagnetic resonance spectrum in the dark state. The dark conformations of five mutant complexes that do not discriminate between orange and near-UV excitation show shifts to lower or higher 2A'(zz) values correlated with the alterations in their motility behavior to one- and two-photon stimuli. These data are interpreted in terms of a model in which the dark complex is populated by two conformers in the wild type, one that inhibits the CheA kinase (A) and the other that activates it (R), shifted in the dark by mutations and shifted in the wild-type SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.

Proton flux across artificial vesicle bilayers

The study of proton conductance across artificial membranes has revealed a surprisingly high permeability for H+, (Pnet H+). A high Pnet H+ is difficult to reconcile with the biological requirement for the maintenance of pH gradients across the plasma membranes of cells, organellar study was undertaken to examine the role played by cholesterol and phospholipid fatty acid side chain composition in determining how well a membrane will function as a barrier to acid. The effects of counter-ion movement on acidification rates were examined in order to interpret the data obtained from variations in membrane composition. In phosphate buffered saline solutions, vesicle membranes composed of unsaturated fatty acid phosphatidylcholines proved to be poorer barriers to acid than membranes composed of saturated fatty acids. The barrier properties of these membranes could be ranked in the following order: DPL, (palmitic) $>$ Egg PC, (mixed chains) $>$ DLL, (linoleic), with DPL being the most effective in maintaining a one pH unit gradient near neutrality. Cholesterol decreased acidification rates of membranes made from the unsaturated phosphatidylcholines Egg PC and DLL, but enhanced acidification rates in vesicle membranes composed of the saturated phospholipid DPL. The cholesterol and fatty acid side chain effects were mediated by changes in membrane fluidity, with more rigid bilayers forming better barriers to acid. Experimental evidence was obtained which confirmed the Pnet H+ is very high relative to the permeabilities of other ions. Counter-ion controlled acidification rates depended on the size and charge of the ion which was moving in order to maintain electroneutrality. The biological relevance of a high intrinsic Pnet H+ and the possible role of counter-ion controlled acidification were discussed. ^

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^

Regulation of p53 antiproliferative function by transactivation domain phosphorylation at threonine 18 and serine 20

We investigated the induction and physiological role of Thr18 and Ser20 phosphorylation of p53 in response to DNA damage caused by treatment with ionizing (IR) or ultraviolet (UV) radiation. Polyclonal antibodies specifically recognizing phospho-Thr18 and phospho-Ser20 were used to detect p53 phosphorylation in vivo. Analyses of five wild-type (wt) p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 and Ser20 after treatment with IR or UV. Importantly, the phosphorylation of p53 at Thr18 and Ser20 correlated with induction of the p53 downstream targets p21Waf1/Cip1 (p21) and Mdm-2, suggesting a transactivation enhancing role for Thr18 and Ser20 phosphorylation. Whereas Thr18 phosphorylation appears to abolish side-chain hydrogen bonding between Thr18 and Asp21, Ser20 phosphorylation may introduce charge attraction between Ser20 and Lys24. Both of these interactions could contribute to stabilizing α-helical conformation within the p53 transactivation domain. Mutagenesis-derived phosphorylation mimicry of p53 at Thr18 and Ser20 by Asp substitution (p53T18D/S20D) altered transactivation domain conformation and significantly reduced the interaction of p53 with the transactivation repressor Mdm-2. Mdm-2 interaction was also reduced with p53 containing a single site Asp substitution at Ser20 (p53S20D) and with the Thr18/Asp21 hydrogen bond disrupting p53 mutants p53T18A, p53T18D and p53D21A. In contrast, no direct effect was observed on the interaction of p53T18A, p53T18D and p53D21A with the basal transcription factor TAF II31. However, prior incubation of p53T18A, p53T18D and p53D21A with Mdm-2 modulated TAFII31 interaction, suggesting Mdm-2 blocks the accessibility of p53 to TAFII31. Consistently, p53-null cells transfected with p53S20D and p53T18A, p53T18D and p53D21A demonstrated enhanced endogenous p21 expression; transfection with p53T18D/S20D most significantly enhanced p21 and fas/APO-1 (fas ) expression. Expression of p53T18A, p53T18D and p53D21A in p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. Cell proliferation was also significantly curtailed in p53-null cells transfected with p53T18D/S20D relative to cells transfected with wt p53. We conclude the irradiation-induced phosphorylation of p53 at Thr18 and Ser20 alters the α-helical conformation of its transactivation domain. Altered conformation reduces direct interaction with the transrepressor Mdm-2, enhancing indirect recruitment of the basal transcription factor TAFII31, facilitating sequence-specific transactivation function resulting in proliferative arrest. ^