Membrane proteins are largely underrepresented among available atomic-resolution structures. The use of detergents in protein purification procedures hinders the formation of well-ordered crystals for X-ray crystallography and leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy is an emerging method for membrane-protein structural biology that can overcome these technical problems. Here we present the solid-state NMR structure of the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA). The sample was derived from crystallization trials that yielded only poorly diffracting microcrystals. We solved the structure using a single, uniformly (13)C- and (15)N-labeled sample. In addition, solid-state NMR allowed us to acquire information on the flexibility and mobility of parts of the structure, which, in combination with evolutionary conservation information, presents new insights into the autotransport mechanism of YadA.

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The assignment of nonexchanging protons of a small microcrystalline protein, the alpha-spectrin SH3 domain (7.2 kDa, 62 residues), was achieved by means of three-dimensional (3D) heteronuclear (1H-13C-13C) magic-angle spinning (MAS) NMR dipolar correlation spectroscopy. With the favorable combination of a high B(0)-field, a moderately high spinning frequency, and frequency-switched Lee-Goldburg irradiation applied during 1H evolution, a proton linewidth < or =0.5 ppm at 17.6 Tesla was achieved for the particular protein preparation used. A comparison of the solid-state 1H chemical shifts with the shifts found in solution shows a remarkable similarity, which reflects the identical protein structures in solution and in the solid. Significant differences between the MAS solid- and liquid-state 1H chemical shifts are only observed for residues that are located at the surface of the protein and that exhibit contacts between different SH3 molecules. In two cases, aromatic residues of neighboring SH3 molecules induce pronounced upfield ring-current shifts for protons in the contact area.

SignificanceUnderstanding the formation and structure of protective bacterial biofilms will help to design and identify antimicrobial strategies. Our experiments with the secreted major biofilm protein TasA characterize on a molecular level in vivo the transition of a folded protein into protease-resistant biofilm-stabilizing fibrils. Such conformational changes from a globular state into fibrillar structures are so far not seen for other biofilm-forming proteins. In this context, TasA can serve as a model system to study functional fibril formation from a globular state.

In this paper, a three-dimensional (3D) NMR-based approach for the determination of the fold of moderately sized proteins by solid-state magic-angle spinning (MAS) NMR is presented and applied to the R-spectrin SH3 domain. This methodology includes the measurement of multiple 13 C- 13 C distance restraints on biosynthetically site-directed 13 C-enriched samples, obtained by growing bacteria on [2-13 C]glycerol and [1,3-13 C]glycerol. 3D 15 N-13 C-13 C dipolar correlation experiments were applied to resolve overlap of signals, in particular in the region where backbone carbon-carbon correlations of the C R -C R , CO-CO, C R -CO, and CO-C R type appear. Additional restraints for confining the structure were obtained from φ and ψ backbone torsion angles of 29 residues derived from C R , C , CO, NH, and H R chemical shifts. Using both distance and angular restraints, a refined structure was calculated with a backbone root-mean-square deviation of 0.7 Å with respect to the average structure.Many biological systems, such as membrane proteins and amyloid fibrils, remain a challenge in structural biology because of difficulties with crystallization and solubility. In the past years, solid-state NMR 1 has become a promising method for obtaining structural information about these systems, via the measurement of accurate distances (1-7), φ and backbone torsion angles (8-10), and chemical shift anisotropy (11,12). In these studies, samples labeled only in the positions of interest were investigated. For the determination of the complete protein folds, however, a different approach that allows the collection of a large number of structural restraints from a small number of samples has to be followed. The quality of the structures increases with the number of restraints, and the more that are measured, the lower the accuracy of the individual restraints may be. From a close analysis of the topology of helical and -sheet structures, it transpires that carboncarbon distances are very important in defining the fold of a protein. For example, distances between backbone carbons, i.e., R-carbons and carbonyl carbons, define the secondary structure of a protein (Table 1). Distances between backbone and side chain carbons or between side chain and side chain carbons provide information about the tertiary structure. The detection of structure-defining long-range carbon-carbon restraints is only possible when so-called dipolar truncation effects are suppressed (13,14). This can be accomplished by employing a reduced labeling scheme, in which chemically bonded carbons are not simultaneously labeled and hence the number of strong dipolar couplings between connected nuclei is reduced. For proteins expressed in bacterial systems, this can be achieved by using [2-13 C]glycerol or [1,3-13 C]glycerol as the only carbon source in the media (15)(16)(17). In combination with this labeling pattern, long-range 13 C-13 C distance restraints may be collected by using a broad-band recoupling method like the proton-driven spindiffusion (PDSD...