H. J. M. de Groot

Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning.

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle ψ previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions.

Asymmetric binding of the 1- and 4-C=O groups of QA in Rhodobacter sphaeroides R26 reaction centres monitored by Fourier transform infra-red spectroscopy using site-specific isotopically labelled ubiquinone-10.

Using 1‐, 2‐, 3‐ and 4‐13C site‐specifically labelled ubiquinone‐10, reconstituted at the QA site of Rhodobacter sphaeroides R26 reaction centres, the infra‐red bands dominated by the 1‐ and 4‐C = O vibration of QA are assigned in the QA(‐)‐QA difference spectra. The mode dominated by the 4‐C = O vibration is drastically downshifted in the reaction centres as compared with its absorption frequency in free ubiquinone‐10. In contrast, the mode dominated by the 1‐C = O vibration absorbs at similar frequencies in the free and the bound forms. The frequency shift of the 4‐C = O vibration is due to a large decrease in bond order and indicates a strong interaction with the protein microenvironment in the ground state. In the charge‐separated state the mode dominated by the semiquinone 4‐C = O vibration is characteristic of strong hydrogen bonding to the microenvironment, whereas the mode dominated by the 1‐C = O vibration indicates a weaker interaction. The asymmetric binding of the 1‐ and 4‐C = O groups to the protein might contribute to the factors governing different redox reactions of ubiquinone‐10 at the QA site as compared with its reactions at the QB site.

Heteronuclear 2D-correlations in a uniformly [13C, 15N] labeled membrane-protein complex at ultra-high magnetic fields.

One- and two-dimensional solid-state NMR experiments on a uniformly labeled intrinsic membrane-protein complex at ultra-high magnetic fields are presented. Two-dimensional backbone and side-chain correlations for a [U-13C,15N] labeled version of the LH2 light-harvesting complex indicate significant resolution at low temperatures and under Magic Angle Spinning. Tentative assignments of some of the observed correlations are presented and attributed to the α-helical segments of the protein, mostly found in the membrane interior.

Determination of a molecular torsional angle in the metarhodopsin-I photointermediate of rhodopsin by double-quantum solid-state NMR

We present a solid-state NMR study of metarhodopsin-I, the pre-discharge intermediate of the photochemical signal transduction cascade of rhodopsin, which is the 41 kDa integral membrane protein that triggers phototransduction in vertebrate rod cells. The H-C10-C11-H torsional angles of the retinylidene chromophore in bovine rhodopsin and metarhodopsin-I were determined simultaneously in the photo-activated membrane-bound state, using double-quantum heteronuclear local field spectroscopy. The torsional angles were estimated to be |φ| = 160 ± 10° for rhodopsin and φ= 180 ± 25° for metarhodopsin-I. The result is consistent with current models of the photo-induced conformational transitions in the chromophore, in which the 11-Z retinal ground state is twisted, while the later photointermediates have a planar all-E conformation.

FTIR spectroscopy shows weak symmetric hydrogen bonding of the QB carbonyl groups in Rhodobacter sphaeroides R26 reaction centres

The absorption frequencies of the C = O and C = C (neutral state) and of the C̲⋯O (semiquinone state) stretching vibrations of QB have been assigned by FTIR spectroscopy, using native and site‐specifically 1‐, 2‐, 3‐ and 4‐13C‐labelled ubiquinone‐10 (UQ10) reconstituted at the QB binding site of Rhodobacter sphaeroides R26 reaction centres. Besides the main C = O band at 1641 cm−1, two smaller bands are observed at 1664 and 1651 cm−1. The smaller bands at 1664 and 1651 cm−1 agree in frequencies with the 1‐ and 4‐C = O vibrations of unbound UQ10, showing that a minor fraction is loosely and symmetrically bound to the protein. The larger band at 1641 cm−1 indicates symmetric H‐bonding of the 1‐ and 4‐C = O groups for the layer fraction of UQ10 but much weaker interaction as for the 4‐C = O group of QA The FTIR experiments show that different C = O protein interactions contribute to the factors determining the different functions of UQ10 at the QA and the QB binding sites.

MAS NMR structure refinement of uniformly 13C enriched chlorophyll a/water aggregates with 2D dipolar correlation spectroscopy

Abstract 2D MAS dipolar correlation spectroscopy of uniformly 13 C enriched chlorophyll a /water aggregates was performed using broadband RFDR to promote exchange of coherence through homonuclear dipolar couplings. It is shown experimentally that incorporation of TPPI and coherence pathway selection in the RFDR pulse scheme yields virtually pure 2D absorption lineshapes. From a series of 2D correlation spectra collected at two spinning speeds with different mixing times of ∼ 1 and ∼ 10 ms all 13 C resonances of the chlorophyll a molecule were assigned. The spectra with the longer mixing times reveal through-space intermolecular polarization transfer. This demonstrates that structural information can be obtained from uniformly 13 C enriched samples, which paves the way for a full structural characterization of amorphous solids.

A physical interpretation of the Floquet description of magic angle spinning nuclear magnetic resonance spectroscopy

A physical interpretation of the Floquet description for magic angle spinning (MAS) nuclear magnetic resonance (NMR) is proposed. The effect of the spatial rotation on the spin system in sample spinning is analysed and described in terms of orbital angular momentum operators. The analogy between rotations in real space and in spin space is emphasized. The transformation properties of the irreducible tensors in real space are used to construct a Floquet Hamiltonian for MAS NMR, that is time independent and comprises one term associated with pure sample rotation. The remaining terms are associated with the spin system, and consist of spin-phonon type Floquet operators generating simultaneous transitions between rotational states and spin states. Finally, two different definitions for the Floquet density operator are compared.

MULTIDIMENSIONAL CP-MAS 13C NMR OF UNIFORMLY ENRICHED CHLOROPHYLL

Abstract The progress toward structure refinement of solid-type uniformly 13C enriched ([U-13C]) chlorophyll-containing biological preparations is summarised. Solid state carbon chemical shifts of aggregated [U-13C] bacteriochlorophyll (BChl) c in intact chlorosomes of Chlorobium tepidum and in [U-13C] BChl c aggregates were determined by the application of homonuclear (13C13C) magic angle spinning (MAS) NMR dipolar correlation spectroscopy. It was found that the arrangement of BChl c molecules in the chlorosomes and in the aggregates is highly similar, which provides convincing evidence that self-organisation of the BChl c is the main mechanism to support the structure of the chlorosomes. Additionally, high field 2-D (1H13C) and 3-D (1H13C13C) dipolar correlation spectroscopy was applied to determine solid state proton chemical shifts of aggregated [U-13C] BChl c in intact chlorosomes. From the high-field assignments, evidence is found for the existence of at least two well-defined interstack arrangements.

Multiple-spin effects in fast magic angle spinning Lee–Goldburg cross-polarization experiments in uniformly labeled compounds

The proton–carbon polarization exchange in Lee–Goldburg cross-polarization magic angle spinning (LG-CP MAS) nuclear magnetic resonance experiments on uniformly 13C-labeled compounds at high spinning frequency is studied. It is shown that the multiple carbon labels in the samples greatly influence the spin dynamics during the LG-CP mixing times. The zeroth order effective LG-CP MAS spin Hamiltonian is a sum of zero quantum dipolar interaction terms. These pairwise dipolar terms generally do not commute with each other, making it impossible to factorize the evolution operator. Consequently, the frequencies of the dipolar oscillations as well as the polarization transfer amplitudes become strongly dependent on the configuration of the spins involved in the multiple heteronuclear couplings. The strong carbon–proton couplings usually attenuate polarization transfers between weakly coupled spins. In practice, this implies that except for strongly coupled or isolated heteronuclear 13C–1H spin pairs, it is diffic...

Protein‐chromophore interactions in α‐crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus a study by 13C magic angle spinning NMR

MAS (magic angle spinning) 13C NMR has been used to study protein‐chromophore interactions in α‐crustacyanin, the blue astaxanthin‐binding carotenoprotein of the lobster, Homarus gammarus, reconstituted with astaxanthins labelled with 13C at the 14,14′ or 15,15′ positions. Two signals are seen for α‐crustacyanin containing [14,14′‐13C2]astaxanthin, shifted 6.9 and 4.0 ppm downfield from the 134.1 ppm signal of uncomplexed astaxanthin in the solid state. With α‐crustacyanin containing [15,15′‐13C2]astaxanthin, one essentially unshifted broad signal is seen. Hence binding to the protein causes a decrease in electronic charge density, providing the first experimental evidence that a charge redistribution mechanism contributes to the bathochromic shift of the astaxanthin in α‐crustacyanin, in agreement with inferences based on resonance Raman data [Salares, et al. (1979) Biochim. Biophys. Acta 576, 176–191]. The splitting of the 14 and 14′ signals provides evidence for asymmetric binding of each astaxanthin molecule by the protein.