Dipolar dephasing for structure determination in a paramagnetic environment.

We demonstrate the efficacy of the REDOR-type sequences in determining dipolar coupling strength in a paramagnetic environment. Utilizing paramagnetic effects of enhanced relaxation rates and rapid electronic fluctuations in Cu(II)-(DL-Ala)2.H2O, the dipolar coupling for the methyl C-H that is 4.20 â€‹Å (methyl carbon) away from the Cu2+ ion, was estimated to be 8.8 â€‹± â€‹0.6 â€‹kHz. This coupling is scaled by a factor of ~0.3 in comparison to the rigid limit value of ~32 â€‹kHz, in line with partial averaging of the dipolar interaction by rotational motion of the methyl group. Limited variation in the scaling factor of the dipolar coupling strength at different temperatures is observed. The C-H internuclear distance derived from the size of the dipolar coupling is similar to that observed in the crystal structure. The errors in the dipolar coupling strength observed in the REDOR-type experiments are similar to those reported for diamagnetic systems. Increase in resolution due to the Fermi contact shifts, coupled with MAS frequencies of 30-35 â€‹kHz allowed to estimate the hyperfine coupling strengths for protons and carbons from the temperature dependence of the chemical shift and obtain a high resolution 1H-1H spin diffusion spectrum. This study shows the utility of REDOR-type sequences in obtaining reliable structural and dynamical information from a paramagnetic complex. We believe that this can help in studying the active site of paramagnetic metalloproteins at high resolution.

The Resting Oxidized State of Small Laccase Analyzed with Paramagnetic NMR Spectroscopy.

The enzyme laccase catalyzes the reduction of dioxygen to water at the trinuclear copper center (TNC). The TNC comprises a type-3 (T3) and a type-2 (T2) copper site. The paramagnetic NMR spectrum of the small laccase from Streptomyces coelicolor (SLAC) without the substrate shows a mixture of two catalytic states, the resting oxidized (RO) state and the native intermediate (NI) state. An analysis of the resonances of the RO state is reported. In this state, hydrogen resonances only of the T3 copper ligands can be found, in the region of 12-22 ppm. Signals from all six histidine ligands are found and can be attributed to Hδ1, Hß or backbone amide HN nuclei. Two sequence-specific assignments are proposed on the basis of a second-coordination shell variant that also lacks the copper ion at the T1 site, SLAC-T1D/Q291E. This double mutant is found to be exclusively in the RO state, revealing a subtle balance between the RO and the NI states.

Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei.

Laccases efficiently reduce dioxygen to water in an active site containing a tri-nuclear copper centre (TNC). The dynamics of the protein matrix is a determining factor in the efficiency in catalysis. To probe mobility, nuclear magnetic resonance (NMR) spectroscopy is highly suitable. However, several factors complicate the assignment of resonances to active site nuclei in laccases. The paramagnetic nature causes large shifts and line broadening. Furthermore, the presence of slow chemical exchange processes of the imidazole rings of copper ligand results in peak doubling. A third complicating factor is that the enzyme occurs in two states, the native intermediate (NI) and resting oxidized (RO) states, with different paramagnetic properties. The present study aims at resolving the complex paramagnetic NMR spectra of the TNC of Streptomyces coelicolor small laccase (SLAC). With a combination of paramagnetically tailored NMR experiments, all eight His Nδ1 and Hδ1 resonances for the NI state are identified, as well as His Hß protons for the RO state. With the help of second-shell mutagenesis, selective resonances are tentatively assigned to the histidine ligands of the copper in the type-2 site. This study demonstrates the utility of the approaches used for the sequence-specific assignment of the paramagnetic NMR spectra of ligands in the TNC that ultimately may lead to a description of the underlying motion.

Chemical Exchange at the Trinuclear Copper Center of Small Laccase from Streptomyces coelicolor.

The trinuclear copper center (TNC) of laccase reduces oxygen to water with very little overpotential. The arrangement of the coppers and ligands in the TNC is known to be from many crystal structures, yet information about possible dynamics of the ligands is absent. Here, we report dynamics at the TNC of small laccase from Streptomyces coelicolor using paramagnetic NMR and electron paramagnetic resonance spectroscopy. Fermi contact-shifted resonances tentatively assigned to histidine Hδ1 display a two-state chemical exchange with exchange rates in the order of 100 s-1. In the electron paramagnetic resonance spectra, at least two forms are observed with different gz-values. It is proposed that the exchange processes reflect the rotational motion of histidine imidazole rings that coordinate the coppers in the TNC.

A Hybrid Solid-State NMR and Electron Microscopy Structure-Determination Protocol for Engineering Advanced para-Crystalline Optical Materials.

Hybrid magic-angle spinning (MAS) NMR spectroscopy and TEM were demonstrated for de novo structure determination of para-crystalline materials with a bioinspired fused naphthalene diimide (NDI)-salphen-phenazine prototype light-harvesting compound. Starting from chiral building blocks with C2 molecular symmetry, the asymmetric unit was determined by MAS NMR spectroscopy, index low-resolution TEM diffraction data, and resolve reflection conditions, and for the first time the ability to determine the space group from reciprocal space data using this hybrid approach was shown. Transfer of molecular C2 symmetry into P2/c packing symmetry provided a connection across length scales to overcome both lack of long-range order and missing diffraction-phase information. Refinement with heteronuclear distance constraints confirmed the racemic P2/c packing that was scaffolded by molecular recognition of salphen zinc in a pseudo-octahedral environment with bromide and with alkyl chains folding along the phenazine. The NDI light-harvesting stacks ran orthogonal to the intermolecular electric dipole moment present in the solid. Finally, the orientation of flexible lamellae on an electrode surface was determined.

Antistaphylococcal Prenylated Acylphoroglucinol and Xanthones from Kielmeyera variabilis.

Bioactivity-guided fractionation of the EtOH extract of the branches of Kielmeyera variabilis led to the isolation of a new acylphoroglucinol (1), which was active against all the MRSA strains tested herein, with pronounced activity against strain EMRSA-16. Compound 1 displayed an MIC of 0.5 mg/L as compared with an MIC of 128 mg/L for the control antibiotic norfloxacin. The structure of the new compound was elucidated by 1D and 2D NMR spectroscopic analysis and mass spectrometry, and experimental and calculated ECD were used to determine the absolute configurations. The compounds ß-sitosterol (2), stigmasterol (3), ergost-5-en-3-ol (4), and osajaxanthone (5) also occurred in the n-hexane fraction. The EtOAc fraction contained nine known xanthones: 3,6-dihydroxy-1,4,8-trimethoxyxanthone (6), 3,5-dihydroxy-4-methoxyxanthone (7), 3,4-dihydroxy-6,8-dimethoxyxanthone (8), 3,4-dihydroxy-2-methoxyxanthone (9), 5-hydroxy-1,3-dimethoxyxanthone (10), 4-hydroxy-2,3-dimethoxyxanthone (11), kielcorin (12), 3-hydroxy-2-methoxyxanthone (13), and 2-hydroxy-1-methoxyxanthone (14), which showed moderate to low activity against the tested MRSA strains.