Residual backbone and side-chain 13C and 15N resonance assignments of the intrinsic transmembrane light-harvesting 2 protein complex by solid-state Magic Angle Spinning NMR spectroscopy.

This study reports the sequence specific chemical shifts assignments for 76 residues of the 94 residues containing monomeric unit of the photosynthetic light-harvesting 2 transmembrane protein complex from Rhodopseudomonas acidophila strain 10050, using Magic Angle Spinning (MAS) NMR in combination with extensive and selective biosynthetic isotope labeling methods. The sequence specific chemical shifts assignment is an essential step for structure determination by MAS NMR. Assignments have been performed on the basis of 2-dimensional proton-driven spin diffusion (13)C-(13)C correlation experiments with mixing times of 20 and 500 ms and band selective (13)C-(15)N correlation spectroscopy on a series of site-specific biosynthetically labeled samples. The decreased line width and the reduced number of correlation signals of the selectively labeled samples with respect to the uniformly labeled samples enable to resolve the narrowly distributed correlation signals of the backbone carbons and nitrogens involved in the long alpha-helical transmembrane segments. Inter-space correlations between nearby residues and between residues and the labeled BChl a cofactors, provided by the (13)C-(13)C correlation experiments using a 500 ms spin diffusion period, are used to arrive at sequence specific chemical shift assignments for many residues in the protein complex. In this way it is demonstrated that MAS NMR methods combined with site-specific biosynthetic isotope labeling can be used for sequence specific assignment of the NMR response of transmembrane proteins.

Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: a model for solid state NMR structure determination of transmembrane proteins.

Partly biosynthetic site-directed isotopically (13)C enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4-(13)C], [1,4-(13)C] and [2,3-(13)C] succinic acid as a precursor in the growth medium. Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual (13)C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors. For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from [1,4-(13)C]-succinic acid or [2,3-(13)C]-succinic labeled media. All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor. Carbonyl carbons in the protein backbone were labeled by [1,4-(13)C]-succinic acid, while the Calpha and Cbeta carbons of the residues were labeled by [2,3 (13)C]-succinic acid. Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium. The pattern labeling yields an increase of the resolution and less spectral crowding. The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for alpha-helical transmembrane protein structures.

Direct determination of the single-ion anisotropy in a one-dimensional magnetic system by high-field EPR spectroscopy; synthesis, EPR, and X-ray structure of NixZn1-x(C2O4)(dmiz)2.

The synthesis, X-ray structure, and EPR measurements of the integer-spin linear-chain antiferromagnet [Ni(ox)(dmiz)2] (where ox = C2O4(2-) and dmiz = 1,2-dimethylimidazole) are presented. The sign and size of the single-ion zero field splitting (Zfs) of the divalent Ni have been determined by high field/high-frequency EPR spectroscopy. The spectra of powder samples of the derivatives [NixZn1-x(C2O4)(dmiz)2] for x = 0.09 and 0.07, at frequencies ranging from 110 to 440 GHz allowed the accurate determination of the zfs parameters D and E, with D = 1.875(4) cm(-1) and E = 0.38 cm(-1). The X-ray structure has been determined from measurements on a single crystal with x = 0.07. Structural parameters are as follows: a = 14.5252(7) A, b = 12.1916(8) A, c = 8.6850(8) A,beta = 97.460(6)degrees in space group C2/c. The zigzag chain contains octahedrally coordinated metal ions with two cis-oriented N-coordinated dmiz ligands and two cis-oriented, tetradentate bridging oxalato(2-) ligands, together resulting in a MN2O4 donor set. The structure was refined to a conventional R value of 0.073 for 1,051 observed reflections. Zn-O distances are 2.167(5) A and Zn-N = 2.098 A. Coordination angles vary for cis angles from 78.4 to 100.7 degrees, with trans angles varying from 163.9 degrees to 165.5 degrees.

[Pt(dien)]2+ migrates intramolecularly from methionine S to imidazole Nepsilon2 in the peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh.

The pH- and time-dependent reaction of [Pt(dien)(H2O)]2+ with the methionine- and histidine-containing peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh at 313 K has been investigated by HPLC and NMR spectroscopy. For both peptides, initial relatively rapid formation of the kinetically favoured methionine S-bound complex is followed by slow intramolecular migration of the [Pt(dien)]2+ fragment to imidazole Nepsilon2 (or, in the case of H-His-Gly-Met-OH, to a much lesser extent to the competing imidazole Ndelta1) of the histidine side chain over a period of 500 h. Time-dependent studies for the pentapeptide at pH 8.0 demonstrate that this isomerization can take place by either direct S-->Nepsilon2 migration or by a two-step mechanism involving initial Nepsilon2 coordination of a second [Pt(dien)]2+ fragment and subsequent cleavage of the orginal Pt-S bond in the resulting dinuclear complex. The rate of kappaS/kappaNepsilon2 isomerization is markedly reduced on lowering the pH to 5.1.