Solid-state NMR Study of the YadA Membrane-Anchor Domain in the Bacterial Outer Membrane.

MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.

Large protein complexes with extreme rotational correlation times investigated in solution by magic-angle-spinning NMR spectroscopy.

We show that large protein complexes can be investigated in solution using magic-angle-spinning (MAS) NMR spectroscopy without the need for sample crystallization or precipitation. In order to efficiently average anisotropic interactions with MAS, the rotational diffusion of the molecule has to be suppressed. This can be readily achieved by lowering the sample temperature and by adding glycerol to the protein solution. The approach is demonstrated using the human small heat shock protein (sHSP) alphaB-Crystallin, which forms oligomeric assemblies of approximately 600 kDa. We suggest this scheme as an approach for overcoming size limitations imposed by overall tumbling in solution-state NMR investigations of large protein complexes.

A 3-D structural model of solid self-assembled chlorophyll a/H(2)O from multispin labeling and MAS NMR 2-D dipolar correlation spectroscopy in high magnetic field.

Magic angle spinning (MAS) NMR with Lee-Goldburg cross-polarization (LG-CP) is used to promote long-range heteronuclear transfer of magnetization and to constrain a structural model for uniformly labeled chlorophyll a/H(2)O. An effective maximum transfer range d(max) can be determined experimentally from the detection of a gradually decreasing series of intramolecular correlations with the (13)C along the molecular skeleton. To probe intermolecular contacts, d(max) can be set to approximately 4.2 A by choosing an LG-CP contact time of 2 ms. Long-range (1)H-(13)C correlations are used in conjunction with carbon and proton aggregation shifts to establish the stacking of the chlorophyll a (Chl a) molecules. First, high-field (14.1 T) 2-D MAS NMR homonuclear ((13)C-(13)C) dipolar correlation spectra provide a complete assignment of the carbon chemical shifts. Second, proton chemical shifts are obtained from (1)H-(13)C heteronuclear dipolar correlation spectroscopy in high magnetic field. The shift constraints and long-range (1)H-(13)C intermolecular correlations reveal a 2-D stacking homologous to the molecular arrangement in crystalline solid ethyl-chlorophyllide a. A doubling of a small subset of the carbon resonances, in the 7-methyl region of the molecule, provides evidence for two marginally different well-defined molecular environments. Evidence is found for the presence of neutral structural water molecules forming a hydrogen-bonded network to stabilize Chl a sheets. In line with the microcrystalline order observed for the rings, the long T(1)'s, and absence of conformational shifts for the (13)C in the phytyl tails, it is proposed that the Chl a form a rigid 3-D space-filling structure. Probably the only way this can be realized with the sheets is by forming bilayers with interpenetration of elongated tails. Such a 3-D space-filling organization of the aggregated Chl a from MAS NMR would match existing models inferred from electron microscopy and low-resolution X-ray powder diffraction, while a micellar model based on neutron diffraction and antiparallel stacking observed in solution can be discarded.

Characterization of (1)H-(1)H distances in a uniformly (2)H,(15)N-labeled SH3 domain by MAS solid-state NMR spectroscopy (section sign).

In this communication, we demonstrate the feasibility of obtaining long-range (1)H-(1)H distance information by MAS solid-state NMR for a microcrystalline, uniformly (2)H,(15)N-labeled sample of a SH3 domain of chicken alpha-spectrin. The experiments yield NOESY-type spectra and rely on the favorable dispersion of the (15)N chemical shifts of the protein backbone. Perdeuteration of nonexchangeable sites is employed to simplify proton spin systems and to obtain multiple structural information. Two mixing schemes, (1)H-(1)H double quantum filtered Post-C7 and (1)H spin diffusion, are implemented to obtain quantitative (1)H-(1)H distance information. Post-C7 and spin diffusion cross-peak buildup rates are discussed for initial-rate fitting and in the framework of n = 0 rotational resonance (rotor driven spin diffusion), respectively. Different deuteration schemes were tested to find conditions where short-range (1)H-(1)H interactions are truncated (e.g., between H(N) and H(alpha)), but long-range interactions are retained (e.g., between H(N) and H(N)).

1H detection in MAS solid-state NMR spectroscopy of biomacromolecules employing pulsed field gradients for residual solvent suppression.

In this communication, we demonstrate the feasibility of 1H detection in MAS solid-state NMR for a microcrystalline, uniformly 2H,15N-labeled sample of a SH3 domain of chicken alpha-spectrin, using pulsed field gradients for suppression of water magnetization. Today, B0 gradients are employed routinely in solution-state NMR for coherence order selection and solvent suppression. We suggest to use gradients to purge water magnetization which cannot be suppressed using conventional water suppression schemes. The achievable gain in sensitivity for 1H detection is in the order of 5 compared to the 15N detected version of the experiment (at a MAS rotation frequency of 13.5 kHz). We expect that this labeling concept which achieves high sensitivity due to 1H detection, in combination with the possibility to measure long range 1H-1H distances as we have shown previously, to be a useful tool for the determination of protein structures in the solid state.