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Permeabilization of the outer mitochondrial membrane is an integral step in apoptosis. The resulting release of pro-apoptotic signaling proteins leads to cell destruction through activation of the cysteine-aspartic protease (caspase) cascade. However, the mechanism of outer mitochondrial membrane (OMM) permeabilization remains unclear. It was recently shown that cytochrome c can induce pore formation in cardiolipin-containing phospholipid membranes, leading to large dextran and protein permeability. In this work, the interaction of cytochrome c with cardiolipin-containing phospholipid vesicles, serving as models of the OMM, is investigated to probe cytochrome c-induced permeability. Lipid vesicles having either a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixed-DPPC/cardiolipin membrane and containing a membrane-impermeable Raman tracer 3-nitrobenzenesulfonate (3-NBS) were optically trapped, translated into a solution containing cytochrome c, and monitored for 3-NBS leakage. Cytochrome-correlated leakage was observed only in cardiolipin-containing vesicles. Structural changes observed in the Raman spectra during permeabilization indicated acyl chain disordering along with decreased intensity of the cardiolipin cis-double-bond stretching modes. When the vesicle-associated cytochrome c Raman spectrum is compared with a spectrum in buffer, heme-resonance bands are absent, indicating loss of Met-80 coordination. To verify selective interactions of cytochrome c with cardiolipin, these experiments were repeated where the DPPC acyl chains were deuterated (D62-DPPC), allowing spectral resolution of the DPPC acyl chain response from that of cardiolipin. Interestingly, D62-DPPC acyl chains were unaffected by cytochrome c accumulation, while cardiolipin showed major changes in acyl chain structure. These results suggest that cytochrome-induced permeabilization proceeds through selective interaction of cytochrome c with cardiolipin, resulting in protein unfolding, where the unfolded form interacts with cardiolipin acyl chains within the bilayer to induce permeability. [ABSTRACT FROM AUTHOR]

Reaction of RuCl2(PPh3)3 with LiNN′ (NN′ = 2-[(2,6-diisopropylphenyl)imino]pyrrolide) affords a single product, with the empirical formula RuCl[(2,6-iPr2C6H3)N=CHC4H3N](PPh3)2. We identify this species as a σ-pyrrolato complex, [Ru(NN′)(PPh3)2]2(μ-Cl)2 (3b), rather than mononuclear RuCl(NN′)(PPh3)2 (3a), on the basis of detailed 1D and 2D NMR characterization in solution and in the solid state. Retention of the chelating, σ-bound iminopyrrolato unit within 3b, despite the presence of labile (dative) chloride and PPh3 donors, indicates that the chelate effect is sufficient to inhibit σ → π isomerization of 3b to a piano-stool, π-pyrrolato structure. 2D COSY, SECSY, and J-resolved solid-state 31p NMR experiments confirm that the PPh3 ligands on each metal center are magnetically and crystallographically inequivalent, and 31p CP/MAS NMR experiments reveal the largest 99Ru-31P spin-spin coupling constant (¹J(99Ru,31P) = 244 ± 20 Hz) yet measured. Finally, 31P dipolar-chemical shift spectroscopy is applied to determine benchmark phosphorus chemical shift tensors for phosphine ligands in hexacoordinate ruthenium complexes. [ABSTRACT FROM AUTHOR]

The results of a detailed systematic chlorine solid-state NMR study of several hydrochloride salts of amino acids implicated in chloride ion transport channel selectivity are reported. 35Cl and 37Cl NMR spectra have been obtained for stationary and/or magic-angle spinning powdered samples of the following compounds on 500 and/or 900 MHz spectrometers: DL-arginine HCI monohydrate, L-lysine HCI, L-serine HCI, L-glutamic acid HCI, L-proline HCI, L-isoleucine HCI, L-valine HCI, L-phenylalanine HCI, and glycine HCI. Spectral analyses provide information on the anisotropic properties and relative orientations of the chlorine electric field gradient and chemical shift (CS) tensors, which are intimately related to the local molecular and electronic structure. Data obtained at 900 MHz provide unique examples of the effects of CS anisotropy on the NMR spectrum of a quadrupolar nucleus. The range of chlorine quadrupolar coupling constants (CQ) measured, -6.42 to 2.03 MHz, demonstrates the sensitivity of this parameter to the chloride ion environment and suggests the applicability of chlorine solid-state NMR as a novel experimental tool for defining chloride binding environments in larger ion channel systems. Salts of hydrophobic amino acids are observed to tend to exhibit larger values of CQ than salts of hydrophilic amino acids. A simple model for rationalizing the observed trend in CQ is proposed. For salts for which neutron diffraction structures are available, we identify a quantum chemical method which reproduces experimental values of CQ with a root-mean-square deviation of 0.1 MHz and a correlation coefficient of 0.9998. On the basis of this, chlorine NMR tensors are predicted for the Cl- binding site in CIC channels. [ABSTRACT FROM AUTHOR]

Incomplete motional averaging of chemical shift anisotropy upon weak alignment of nucleic acids and proteins in a magnetic field results in small changes in chemical shift. Knowledge of nucleus-specific chemical shift (CS) tensor magnitudes and orientations is necessary to take full advantage of these measurements in biomolecular structure determination. We report the determination by liquid crystal NMR of the CS tensors for all ribose carbons in A-form helical RNA, using a series of novel 3D NMR pulse sequences for accurate and resolved measurement of the ribose 13C chemical shifts. The orientation of the riboses relative to the rhombic alignment tensor of the molecule studied, a stem-loop sequence corresponding to helix-35 of 238 rRNA, is known from an extensive set of residual dipolar couplings (RDC), previously used to refine its structure. Singular-value-decomposition fits of the chemical shift changes to this structure, or alternatively to a database of helical RNA X-ray structures, provide the CS tensor for each type of carbon. Quantum chemical calculations complement the experimental results and confirm that the most shielded tensor component lies approximately along the local carbon-oxygen bond axis in all cases and that shielding anisotropy for C3' and C4' is much larger than for C1' and C2', with C5' being intermediate. [ABSTRACT FROM AUTHOR]