Structural changes in photosystem II after treatment with the zero-length bifunctional cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide: an electron microscopic study.
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ABSTRACT:
SUBMITTER: Collins RF
PROVIDER: S-EPMC1217807 | biostudies-other | 1996 Oct
REPOSITORIES: biostudies-other
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Project description:The reaction kinetics of native and carbodi-imide-modified tuna and horse heart cytochromes c with both a strong (dithionite) and a relatively weak (ascorbate) reducing agent were studied over a wide range of conditions. In their reactions with dithionite both the native and modified cytochromes exhibit single exponential time courses. The effects of dithionite concentration and ionic strength on the rate of the reduction are complex and can best be explained in terms of the model proposed by Lambeth & Palmer [(1973) J. Biol. Chem. 248, 6095-6103]. According to this model, at low ionic strength the native proteins are reduced almost exclusively by S2O4(2-) whereas the modified proteins showed reactivity towards both S2O4(2-) and SO2.-. These findings are interpreted in terms of the different charge characteristics of the carbodi-imide-modified proteins relative to the native proteins. The findings that the modified proteins react with ascorbate in a biphasic manner are explained as arising from ascorbate binding to a reducible form of the protein, before electron transfer, with an equilibrium between the ascorbate-reducible form of the protein and a non-reducible form. Estimates were obtained for both the ascorbate equilibrium binding constant and the rate constant for the internal electron transfer for both the native and modified horse and tuna proteins. The effect of pH on the reactions indicates that the active reductant in all cases is ascorbate2-. The studies of ascorbate reactivity yield important information concerning the proposed correlation between ascorbate reducibility and the presence of a 695 nm-absorption band, and the study of dithionite reactivity illustrates the effect of protein charge and solution ionic strength on the relative contributions made by the species SO2.- and S2O4(2-) to the reduction of ferricytochrome c.
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Project description:Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11 +/- 4 and 96 +/- 16 microM after 30 min at 22 degrees C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and K(m) for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 degrees C respectively. Spermine (10 microM) almost fully protected against spermidine-transport inactivation by EDC at 22 degrees C, and decreased the rate of inactivation at 37 degrees C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 degrees C with EC50 values estimated at 10, 1 and less than 1 microM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89 +/- 3 compared with 44 +/- 5%), whereas the converse was true for 100 microM DCC (81 +/- 3 compared with 92 +/- 5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na(+)-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to crosslink a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.
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Project description:The reactivity of carbodi-imide-modified tuna and horse heart cytochromes c with the ferrous ion ligands CO and O2 has been studied. Both modified cytochromes bind one molecule of CO. Stopped-flow and flash-photolysis experiments indicate the presence of three kinetic processes in the reaction of the cytochromes with CO. The second-order rate constants associated with all three kinetic process are pH-independent being 2.8 x 10(5) M-1.s-1, 3.8 x 10(4) M-1.s-1 and 4 x 10(3) M-1.s-1 under all conditions studied. The concentration-dependence of the contributions made by each of the processes to the overall absorbance change indicates that the fast and slow kinetic phases are associated with two forms of the cytochromes which are in equilibrium, whereas the intermediate phase arises from a separate cytochrome species. The quantum yield for the photodissociation of CO from the ferrous cytochromes is unusually low. Both modified cytochromes are capable of binding and reducing O2. In the presence of excess reductant, the modified cytochromes can catalytically reduce large molar excesses of O2. In the absence of excess reducing agent, the oxy complex initially formed undergoes a pH-dependent intramolecular electron-transfer process with half-life approx. 10 min. EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide]-promoted internal cross-linking is proposed to account for differences between the EDC-modified proteins and carboxymethylated cytochrome c.
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Project description:There are two independent mol-ecules in the asymmetric unit of the title compound, C16H15NO3S2, in which the pyrrolidine rings adopt envelope conformations, with a methyl-ene C atom as the flap. The dihedral angles betweeen the near-planar 2H-chromene ring systems [maximum deviations = 0.0167?(20) and 0.0136?(19)?Å] and the pyrrolidine rings (all atoms) are 83.83?(11) and 82.43?(11)°. In the crystal, inversion dimers linked by pairs of C-H?O hydrogen bonds occur for one of the mol-ecules. Further C-H?O links involving both mol-ecules generate a three-dimensional network.
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Project description:Based on the mechanism of neuropathic pain induction, a new type of bifunctional hybrid peptidomimetics was obtained for potential use in this type of pain. Hybrids consist of two types of pharmacophores that are connected by different types of linkers. The first pharmacophore is an opioid agonist, and the second pharmacophore is an antagonist of the pronociceptive system, i.e., an antagonist of the melanocortin-4 receptor. The results of tests in acute and neuropathic pain models of the obtained compounds have shown that the type of linker used to connect pharmacophores had an effect on antinociceptive activity. Peptidomimetics containing longer flexible linkers were very effective at low doses in the neuropathic pain model. To elucidate the effect of linker lengths, two hybrids showing very high activity and two hybrids with lower activity were further tested for affinity for opioid (mu, delta) and melanocortin-4 receptors. Their complexes with the target receptors were also studied by molecular modelling. Our results do not show a simple relationship between linker length and affinity for particular receptor types but suggest that activity in neuropathic pain is related to a proper balance of receptor affinity rather than maximum binding to any or all of the target receptors.
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Project description:The charge density (CD) distribution of an atom is the difference per unit volume between the positive charge of its nucleus and the distribution of the negative charges carried by the electrons that are associated with it. The CDs of the atoms in macromolecules are responsible for their electrostatic potential (ESP) distributions, which can now be visualized using cryo-electron microscopy at high resolution. CD maps can be recovered from experimental ESP density maps using the negative Laplacian operation. CD maps are easier to interpret than ESP maps because they are less sensitive to long-range electrostatic effects. An ESP-to-CD conversion involves multiplication of amplitudes of structure factors as Fourier transforms of these maps in reciprocal space by 1/d2 , where d is the resolution of reflections. In principle, it should be possible to determine the charges carried by the individual atoms in macromolecules by comparing experimental CD maps with experimental ESP maps.
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Project description:Knowledge of three-dimensional (3D) structures of each individual particles of asymmetric and flexible proteins is essential in understanding those proteins' functions; but their structures are difficult to determine. Electron tomography (ET) provides a tool for imaging a single and unique biological object from a series of tilted angles, but it is challenging to image a single protein for three-dimensional (3D) reconstruction due to the imperfect mechanical control capability of the specimen goniometer under both a medium to high magnification (approximately 50,000-160,000×) and an optimized beam coherence condition. Here, we report a fully mechanical control method for automating ET data acquisition without using beam tilt/shift processes. This method could reduce the accumulation of beam tilt/shift that used to compensate the error from the mechanical control, but downgraded the beam coherence. Our method was developed by minimizing the error of the target object center during the tilting process through a closed-loop proportional-integral (PI) control algorithm. The validations by both negative staining (NS) and cryo-electron microscopy (cryo-EM) suggest that this method has a comparable capability to other ET methods in tracking target proteins while maintaining optimized beam coherence conditions for imaging.
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