Localisation of methionine residues in bacteriorhodopsin by carbonyl 13C-NMR with sequence-specific assignments

AbstractHigh-resolution 13C-NMR experiments have been performed on bacteriorhodopsin biosynthetically labeled with carbonyl-13C amino acids and solubilized in the detergent dodecylmaltoside. 13C-NMR spectra showing good resolution were obtained in the case of labeled amino acids moderately represented in the BR sequence. For BR labeled with [13C]carbonyl methionine, several sequence-specific assignment could be performed by co-labeling with 15N amino acids or proteolysis. These assignments were used to obtain structural data on BR. Water-exposure of methionine side chains in the protein was assessed by studying, using NMR, their oxidation by hydrogen peroxide. Local secondary structure at the level of methionine residues was monitored through the effect of 1H-2H exchange on NMR spectra. It was concluded that Met32, Met68 and Met163 are peripheral while all 6 other methionine residues are deeply embedded within hydrophobic α-helices. These results confirm the current model of the BR folding and secondary structure

Methods for sequential resonance assignment in solid, uniformly 13C, 15N labelled peptides: Quantification and application to antamanide

The application of adiabatic polarization-transfer experiments to resonance assignment in solid, uniformly 13C-15N-labelled polypeptides is demonstrated for the cyclic decapeptide antamanide. A homonuclear correlation experiment employing the DREAM sequence for adiabatic dipolar transfer yields a complete assignment of the Cα and aliphatic side-chain 13C resonances to amino acid types. The same information can be obtained from a TOBSY experiment using the recently introduced P91 12 TOBSY sequence, which employs the J couplings as a transfer mechanism. A comparison of the two methods is presented. Except for some aromatic phenylalanine resonances, a complete sequence-specific assignment of the 13C and 15N resonances in antamanide is achieved by a series of selective or broadband adiabatic triple-resonance experiments. Heteronuclear transfer by adiabatic-passage Hartmann-Hahn cross polarization is combined with adiabatic homonuclear transfer by the DREAM and rotational-resonance tickling sequences into two- and three-dimensional experiments. The performance of these experiments is evaluated quantitativel

Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase: probing nucleotide binding by multidimensional NMR

ABSTRACT: The skeletal muscle sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1a) mediates muscle relaxation by pumping Ca 2+ from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a