One of the distinctive features of eubacterial retinal based proton pumps, proteorhodopsins, xanthorhodopsin and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in E. coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH dependent properties of ESR. In the H57M mutant a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum upon raising the pH from 5 to 8 indicates deprotonation of the counterion with a pKa of 6.3, which is also the pKa at which the M intermediate is observed in the photocycle of the protein solubilized in detergent (DDM). This is in contrast with the wild type protein, in which the same experiments show that the major fraction of Asp85 is deprotonated at pH > 3 and that it protonates only at low pH, with a pKa of 2.3. The M intermediate in the wild type photocycle accumulates only at high pH, with an apparent pKa of 9 from deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR the pKas for M formation and spectral shifts are 2–3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild type protein vs. the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pKa of Asp85 by stabilizing its deprotonated state.

DNA accessibility to regulatory proteins is significantly affected by nucleosome structure and dynamics. FACT (facilitates chromatin transcription) increases the accessibility of nucleosomal DNA but the mechanism and extent of this nucleosome reorganization are unknown. We report here the effects of FACT on single nucleosomes revealed with spFRET microscopy. FACT binding results in a dramatic, ATP-independent, and reversible uncoiling of DNA that affects at least 70% of the DNA in a nucleosome. A mutated version of FACT is defective in this uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this is an important function of FACT in vivo.

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake ␣-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and threefinger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 M, ws-LYNX1 competed with 125 I-␣-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing ␣7 nAChRs to 1 M ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on ␣4␤2 and ␣3␤2 nAChRs. Increasing ws-LYNX1 concentration to 10 M caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.Endogenous "prototoxins" like LYNX1, LYNX2, SLURP-1, and SLURP-2, belonging to the Ly6 protein family, modulate nicotinic acetylcholine receptors (nAChRs) 3 (1-8). In the central nervous system, LYNX1 and LYNX2 regulate nAChR activity, preventing excessive excitation (3, 4). Gene deletion of LYNX1 or LYNX2 indicates that these modulators are critical for nAChR function in the brain. LYNX1 knock-out mice demonstrated enhanced performance in specific tests of learning ability and memory, whereas loss of LYNX2 results in increased anxiety-related behaviors (3, 4). Prototoxins have also been shown to affect cell growth in lung carcinoma (9), are involved in skin diseases (6, 7), and are related to prostate stem cell antigen (10).LYNX1 and LYNX2 are tethered to the membrane by a GPI anchor, which considerably complicates in vitro studies. LYNX1 is co-localized in the brain with ␣4␤2 and ␣7 nAChRs (1-3), and its modulatory activity on ␣4␤2 nAChR was shown in experiments on Xenopus oocytes (1, 3). It was reported that soluble form of LYNX1 (not containing a GPI anchor) potentiates ␣4␤2 receptor (1), but the concentration at which it acts remains unknown. A secreted water-soluble protein SLURP-1 expressed in palmoplantar skin acts on ␣7 nAChR and regulates keratinocyte proliferation (5).It was predicted that the prototoxins should have a spatial structure similar to that of snake venom ␣-neurotoxins, effective competitive inhibitors of nAChR (1). ␣-Neurotoxins are characterized by a three-finger fold formed by three adjacent loops arising from a small globular hydrophobic core, crosslinked by four conserved disulfide bonds (11-13). Nicotinic acetylcholine receptors are ta...