UV irradiation of polycyclic aromatic hydrocarbons in ices: production of alcohols, quinones, and ethers.
(1/1435)
Polycyclic aromatic hydrocarbons (PAHs) in water ice were exposed to ultraviolet (UV) radiation under astrophysical conditions, and the products were analyzed by infrared spectroscopy and mass spectrometry. Peripheral carbon atoms were oxidized, producing aromatic alcohols, ketones, and ethers, and reduced, producing partially hydrogenated aromatic hydrocarbons, molecules that account for the interstellar 3.4-micrometer emission feature. These classes of compounds are all present in carbonaceous meteorites. Hydrogen and deuterium atoms exchange readily between the PAHs and the ice, which may explain the deuterium enrichments found in certain meteoritic molecules. This work has important implications for extraterrestrial organics in biogenesis. (+info)
Subunit dissociation in fish hemoglobins.
(2/1435)
The tetramer-dimer dissociation equilibria (K 4,2) of several fish hemoglobins have been examined by sedimentation velocity measurements with a scanner-computer system for the ultracentrifuge and by flash photolysis measurements using rapid kinetic methods. Samples studied in detail included hemoglobins from a marine teleost, Brevoortia tyrannus (common name, menhaden); a fresh water teleost, Cyprinus carpio, (common name, carp); and an elasmobranch Prionace glauca (common name, blue shark). For all three species in the CO form at pH 7, in 0.1 M phosphate buffer, sedimentation coefficients of 4.3 S (typical of tetrameric hemoglobin) are observed in the micromolar concentration range. In contrast, mammalian hemoglobins dissociate appreciably to dimers under these conditions. The inability to detect dissociation in three fish hemoglobins at the lowest concentrations examined indicates that K 4,2 must have a value of 10(-8) M or less. In flash photolysis experiments on very dilute solutions in long path length cells, two kinetic components were detected with their proportions varying as expected for an equilibrium between tetramers (the slower component) and dimers (the faster component); values of K 4,2 for the three fish hemoglobins in the range 10(-9) to 10(-8) M were calculated from these data. Thus, the values of K 4,2 for liganded forms of the fish hemoglobins appear to be midway between the value for liganded human hemoglobin (K 4,2 approximately 10(-6) M) and unliganded human hemoglobin (K 4,2 approximately 10(-12) M). This conclusion is supported by measurements on solutions containing guanidine hydrochloride to enhance the degree of dissociation. All three fish hemoglobins are appreciably dissociated at guanidine concentrations of about 0.8 M, which is roughly midway between the guanidine concentrations needed to cause comparable dissociation of liganded human hemoglobin (about 0.4 M) and unliganded human hemoglobin (about 1.6 M). Kinetic measurements on solutions containing guanidine hydrochloride indicated that there are changes in both the absolute rates and the proportions of the fast and slow components, which along with other factors complicated the analysis of the data in terms of dissociation constants. Measurements were also made in solutions containing urea to promote dissociation, but with this agent very high concentrations (about 6 M) were required to give measureable dissociation and the fish hemoglobins were unstable under these conditions, with appreciable loss of absorbance spectra in both the sedimentation and kinetic experiments. (+info)
Selective induction of LTP and LTD by postsynaptic [Ca2+]i elevation.
(3/1435)
Long-term potentiation (LTP) and long-term depression (LTD), two prominent forms of synaptic plasticity at glutamatergic afferents to CA1 hippocampal pyramidal cells, are both triggered by the elevation of postsynaptic intracellular calcium concentration ([Ca2+]i). To understand how one signaling molecule can be responsible for triggering two opposing forms of synaptic modulation, different postsynaptic [Ca2+]i elevation patterns were generated by a new caged calcium compound nitrophenyl-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in CA1 pyramidal cells. We found that specific patterns of [Ca2+]i elevation selectively activate LTP or LTD. In particular, only LTP was triggered by a brief increase of [Ca2+]i with relatively high magnitude, which mimics the [Ca2+]i rise during electrical stimulation typically used to induce LTP. In contrast, a prolonged modest rise of [Ca2+]i reliably induced LTD. An important implication of the results is that both the amplitude and the duration of an intracellular chemical signal can carry significant biological information. (+info)
Structural dynamics of ligand diffusion in the protein matrix: A study on a new myoglobin mutant Y(B10) Q(E7) R(E10).
(4/1435)
A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) --> Tyr, His(E7) --> Gln, and Thr(E10) --> Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O2, and CO from within the protein (in a "geminate" mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O2 binding are reduced 10-fold. While there is no geminate recombination with O2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major "secondary site," located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the "primary site" identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O2 delivery to tissue at a rate compatible with physiology. (+info)
Chemotactic responses of Escherichia coli to small jumps of photoreleased L-aspartate.
(5/1435)
Computer-assisted motion analysis coupled to flash photolysis of caged chemoeffectors provides a means for time-resolved analysis of bacterial chemotaxis. Escherichia coli taxis toward the amino acid attractant L-aspartate is mediated by the Tar receptor. The physiology of this response, as well as Tar structure and biochemistry, has been studied extensively. The beta-2, 6-dinitrobenzyl ester of L-aspartic acid and the 1-(2-nitrophenyl)ethyl ether of 8-hydroxypyrene-1,3,6-tris-sulfonic acid were synthesized. These compounds liberated L-aspartate and the fluorophore 8-hydroxypyrene 1,3,6-tris-sulfonic acid (pyranine) upon irradiation with near-UV light. Photorelease of the fluorophore was used to define the amplitude and temporal stability of the aspartate jumps employed in chemotaxis experiments. The dependence of chemotactic adaptation times on aspartate concentration, determined in mixing experiments, was best fit by two Tar aspartate-binding sites. Signal processing (excitation) times, amplitudes, and adaptive recovery of responses elicited by aspartate jumps producing less than 20% change in receptor occupancy were characterized in photorelease assays. Aspartate concentration jumps in the nanomolar range elicited measurable responses. The response threshold and sensitivity of swimming bacteria matched those of bacteria tethered to glass by a single flagellum. Stimuli of similar magnitude, delivered either by rapid mixing or photorelease, evoked responses of similar strength, as assessed by recovery time measurements. These times remained proportional to change in receptor occupancy close to threshold, irrespective of prior occupancy. Motor excitation responses decayed exponentially with time. Rates of excitation responses near threshold ranged from 2 to 7 s-1. These values are consistent with control of excitation signaling by decay of phosphorylated pools of the response regulator protein, CheY. Excitation response rates increased slightly with stimulus size up to values limited by the instrumentation; the most rapid was measured to be 16 +/- 3 (SE) s-1. This increase may reflect simultaneous activation of CheY dephosphorylation, together with inhibition of its phosphorylation. (+info)
Photolytically released nitric oxide produces a delayed but persistent suppression of LTP in area CA1 of the rat hippocampal slice.
(6/1435)
1. We have used flash photolysis of a caged form of nitric oxide (NO), potassium pentachloronitrosylruthenate (K2Ru(NO)Cl5), to apply known concentrations of NO, with a high degree of temporal resolution, to hippocampal slices prepared from juvenile male rats maintained in an interface recording chamber. 2. Photolytically released NO (1-4.5 microM) from bath applied caged NO reduced the magnitude of long-term potentiation (LTP) in a concentration-dependent manner. This effect was abolished in the presence of the NO scavenger haemoglobin. NO had no effect on pre-established LTP. 3. Exposure to photolytically released NO had no effect on normal fast synaptic transmission, but did result in depression of N-methyl-D-aspartate (NMDA) receptor-mediated transmission recorded using extracellular electrodes. The onset of NO-induced depression was relatively slow, taking >40 s to manifest itself, and several minutes to achieve maximum depression (t approximately 70 s). NO-induced depression persisted for more than 2 h after photolysis. The time courses of the action of NO on NMDA receptor-mediated responses and its action on the induction of LTP were similar. 4. These results suggest that released NO may play a role in determining the subsequent threshold for the induction of LTP at Schaffer-commissural synapses through a reduction in the efficacy of NMDA receptor function when repeated conditioning trains are used. (+info)
Altered ligand rebinding kinetics due to distal-side effects in hemoglobin chico (Lysbeta66(E10) --> thr).
(7/1435)
Hb Chico is an unusual human hemoglobin variant that has lowered oxygen affinity, but unaltered cooperativity and anion sensitivity. Previous studies showed these features to be associated with distal-side heme pocket alterations that confer increased structural rigidity on the molecule and that increase water content in the beta-chain heme pocket. We report here that the extent of nanosecond geminate rebinding of oxygen to the variant and its isolated beta-chains is appreciably decreased. Structural alterations in this variant decrease its oxygen recombination rates without significantly altering rates of migration out of the heme pocket. Data analysis indicates that one or more barriers that impede rebinding of oxygen from docking sites in the heme pocket are increased, with less consequence for CO rebinding. Resonance Raman spectra show no significant alterations in spectral regions sensitive to interactions between the heme iron and the proximal histidine residue, confirming that the functional differences in the variant are due to distal-side heme pocket alterations. These effects are discussed in the context of a schematic representation of heme pocket wells and barriers that could aid the design of novel hemoglobins with altered ligand affinity without loss of the normal allosteric responses that facilitate unloading of oxygen to respiring tissues. (+info)
Time-resolved absorption and photothermal measurements with sensory rhodopsin I from Halobacterium salinarum.
(8/1435)
An expansion accompanying the formation of the first intermediate in the photocycle of transducer-free sensory rhodopsin I (SRI) was determined by means of time-resolved laser-induced optoacoustic spectroscopy. For the native protein (SRI-WT), the absolute value of the expansion is approximately 5.5 mL and for the mutant SRI-D76N, approximately 1.5 mL per mol of phototransformed species (in 0.5 M NaCl), calculated by using the formation quantum yield for the first intermediate (S610) of Phi610 = 0.4 +/- 0.05 for SRI-WT and 0.5 +/- 0.05 for SRI-D76N, measured by laser-induced optoacoustic spectroscopy and by laser flash photolysis. The similarity in Phi610 and in the determined value of the energy level of S610, E610 = (142 +/- 12) kJ/mol for SRI-WT and SRI-D76N indicates that Asp76 is not directly involved in the first step of the phototransformation. The increase with pH of the magnitude of the structural volume change for the formation of S610 in SRI-WT and in SRI-D76N upon excitation with 580 nm indicates also that amino acids other than Asp76, and other than those related to the Schiff base, are involved in the process. The difference in structural volume changes as well as differences in the activation parameters for the S610 decay should be attributed to differences in the rigidity of the cavity surrounding the chromophore. Except for the decay of the first intermediate, which is faster than in the SRI-transducer complex, the rate constants of the photocycle for transducer-free SRI in detergent suspension are strongly retarded with respect to wild-type membranes (this comparison should be done with great care because the preparation of both samples is very different). (+info)