We introduce an improved variant of the C7 pulse-sequence for efficient recoupling of spin-1/2 pair dipolar interactions in magic-angle spinning solid-state NMR spectroscopy. The tolerance of C7 toward isotropic as well as anisotropic chemical shift offsets and rf inhomogeneity is improved considerably by replacing the original basic element Cφ44̄=(2π)φ(2π)φ+π with the cyclically permuted element Cφ14̄3=(π/2)φ(2π)φ+π(3π/2)φ. The improved performance of this permutationally offset stabilized variant of C7 is analyzed by average Hamiltonian theory to fifth order, numerical simulations, and demonstrated by experiments on powder samples of doubly 13C-labeled barium oxalate hemihydrate and diammonium fumarate.

We describe a new fivefold symmetric approach to homonuclear recoupling in rotating solids that is based on rotor-synchronized, spin-lock rf irradiation of the type employed previously in MELODRAMA and C7 ͑and their derivative sequences͒ for 13 C-13 C recoupling. The fivefold sequence, like its sevenfold relatives, is ␥-encoded, and therefore exhibits a theoretical efficiency of ϳ73% for double quantum filtering ͑2QF͒. However, since the ratio of rf field strength, rf /2, to spinning frequency, r /2, is lower, it is possible to operate the sequence at higher spinning rates, and we have investigated the 2QF efficiency as a function of 1 H decoupling field strength at high spinning frequencies. We observe dramatic oscillations of the recoupled signal with a period ϳ r /2 indicating that the 1 H reservoir is behaving partially inhomogeneously. This kind of double quantum recoupling is explored in multiple spin systems and we derive analytical forms for polarization transfer and double quantum excitation relevant for uniformly labeled systems. Finally, the wide applicability of the fivefold sequence is demonstrated with INADEQUATE type spectra of uniformly 13 C labeled sucrose and L-alanine.

The three-dimensional structure of the chemotactic peptide Nformyl-L-Met-L-Leu-L-Phe-OH was determined by using solid-state NMR (SSNMR). The set of SSNMR data consisted of 16 13 C-15 N distances and 18 torsion angle constraints (on 10 angles), recorded from uniformly 13 C, 15 N-and 15 N-labeled samples. The peptide's structure was calculated by means of simulated annealing and a newly developed protocol that ensures that all of conformational space, consistent with the structural constraints, is searched completely. The result is a high-quality structure of a molecule that has thus far not been amenable to single-crystal diffraction studies. The extensions of the SSNMR techniques and computational methods to larger systems appear promising.

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.

We demonstrate constraint of peptide backbone and side-chain conformation with 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift, magic-angle spinning NMR experiments. In these experiments, polarization is transferred from (15)N[i] by ramped SPECIFIC cross polarization to the (13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1] resonances and evolves coherently under the correlated (1)H-(15)N and (1)H-(13)C dipolar couplings. The resulting set of frequency-labeled (15)N(1)H-(13)C(1)H dipolar spectra depend strongly upon the molecular torsion angles phi[i], chi1[i], and psi[i - 1]. To interpret the data with high precision, we considered the effects of weakly coupled protons and differential relaxation of proton coherences via an average Liouvillian theory formalism for multispin clusters and employed average Hamiltonian theory to describe the transfer of (15)N polarization to three coupled (13)C spins ((13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1]). Degeneracies in the conformational solution space were minimized by combining data from multiple (15)N(1)H-(13)C(1)H line shapes and analogous data from other 3D (1)H-(13)C(alpha)-(13)C(beta)-(1)H (chi1), (15)N-(13)C(alpha)-(13)C'-(15)N (psi), and (1)H-(15)N[i]-(15)N[i + 1]-(1)H (phi, psi) experiments. The method is demonstrated here with studies of the uniformly (13)C,(15)N-labeled solid tripeptide N-formyl-Met-Leu-Phe-OH, where the combined data constrains a total of eight torsion angles (three phi, three chi1, and two psi): phi(Met) = -146 degrees, psi(Met) = 159 degrees, chi1(Met) = -85 degrees, phi(Leu) = -90 degrees, psi(Leu) = -40 degrees, chi1(Leu) = -59 degrees, phi(Phe) = -166 degrees, and chi1(Phe) = 56 degrees. The high sensitivity and dynamic range of the 3D experiments and the data analysis methods provided here will permit immediate application to larger peptides and proteins when sufficient resolution is available in the (15)N-(13)C chemical shift correlation spectra.