Experimental Protein Structure Verification by Scoring with a Single, Unassigned NMR Spectrum.

Standard methods for de novo protein structure determination by nuclear magnetic resonance (NMR) require time-consuming data collection and interpretation efforts. Here we present a qualitatively distinct and novel approach, called Comparative, Objective Measurement of Protein Architectures by Scoring Shifts (COMPASS), which identifies the best structures from a set of structural models by numerical comparison with a single, unassigned 2D (13)C-(13)C NMR spectrum containing backbone and side-chain aliphatic signals. COMPASS does not require resonance assignments. It is particularly well suited for interpretation of magic-angle spinning solid-state NMR spectra, but also applicable to solution NMR spectra. We demonstrate COMPASS with experimental data from four proteins--GB1, ubiquitin, DsbA, and the extracellular domain of human tissue factor--and with reconstructed spectra from 11 additional proteins. For all these proteins, with molecular mass up to 25 kDa, COMPASS distinguished the correct fold, most often within 1.5 Å root-mean-square deviation of the reference structure.

Solid-state NMR study of a 41 kDa membrane protein complex DsbA/DsbB.

The disulfide bond generation system in E. coli is led by a periplasmic protein, DsbA, and an integral membrane protein, DsbB. Here we present a solid-state NMR (SSNMR) study of a 41 kDa membrane protein complex DsbA/DsbB precipitated in the presence of native lipids to investigate conformational changes and dynamics that occur upon transient complex formation within the electron transfer pathway. Chemical shift changes in the periplasmic enzyme DsbA in three states (wild type, C33S mutant, and in complex with DsbB) reveal structural and/or dynamic information. We report a 4.9 ppm (15)N chemical shift change observed for Pro31 in the active site between the wild type and C33S mutant of DsbA. Additionally, the Pro31 residue remains elusive in the DsbA/DsbB complex, indicating that the dynamics change drastically in the active site between the three states of DsbA. Using three-dimensional SSNMR spectra, partial (13)C and (15)N de novo chemical shift assignments throughout DsbA in the DsbA/DsbB complex were compared with the shifts from DsbA alone to map site-specific chemical shift perturbations. These results demonstrate that there are further structural and dynamic changes of DsbA in the native membrane observed by SSNMR, beyond the differences between the crystal structures of DsbA and the DsbA/DsbB complex.

Structure of the disulfide bond generating membrane protein DsbB in the lipid bilayer.

The integral membrane protein DsbB in Escherichia coli is responsible for oxidizing the periplasmic protein DsbA, which forms disulfide bonds in substrate proteins. We have developed a high-resolution structural model by combining experimental X-ray and solid-state NMR with molecular dynamics (MD) simulations. We embedded the high-resolution DsbB structure, derived from the joint calculation with X-ray reflections and solid-state NMR restraints, into the lipid bilayer and performed MD simulations to provide a mechanistic view of DsbB function in the membrane. Further, we revealed the membrane topology of DsbB by selective proton spin diffusion experiments, which directly probe the correlations of DsbB with water and lipid acyl chains. NMR data also support the model of a flexible periplasmic loop and an interhelical hydrogen bond between Glu26 and Tyr153.

VITAL NMR: using chemical shift derived secondary structure information for a limited set of amino acids to assess homology model accuracy.

Homology modeling is a powerful tool for predicting protein structures, whose success depends on obtaining a reasonable alignment between a given structural template and the protein sequence being analyzed. In order to leverage greater predictive power for proteins with few structural templates, we have developed a method to rank homology models based upon their compliance to secondary structure derived from experimental solid-state NMR (SSNMR) data. Such data is obtainable in a rapid manner by simple SSNMR experiments (e.g., (13)C-(13)C 2D correlation spectra). To test our homology model scoring procedure for various amino acid labeling schemes, we generated a library of 7,474 homology models for 22 protein targets culled from the TALOS+/SPARTA+ training set of protein structures. Using subsets of amino acids that are plausibly assigned by SSNMR, we discovered that pairs of the residues Val, Ile, Thr, Ala and Leu (VITAL) emulate an ideal dataset where all residues are site specifically assigned. Scoring the models with a predicted VITAL site-specific dataset and calculating secondary structure with the Chemical Shift Index resulted in a Pearson correlation coefficient (-0.75) commensurate to the control (-0.77), where secondary structure was scored site specifically for all amino acids (ALL 20) using STRIDE. This method promises to accelerate structure procurement by SSNMR for proteins with unknown folds through guiding the selection of remotely homologous protein templates and assessing model quality.

High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data.

X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) are the staple methods for revealing atomic structures of proteins. Since crystals of biomolecular assemblies and membrane proteins often diffract weakly and such large systems encroach upon the molecular tumbling limit of solution NMR, new methods are essential to extend structures of such systems to high resolution. Here we present a method that incorporates solid-state NMR restraints alongside of X-ray reflections to the conventional model building and refinement steps of structure calculations. Using the 3.7 Å crystal structure of the integral membrane protein complex DsbB-DsbA as a test case yielded a significantly improved backbone precision of 0.92 Å in the transmembrane region, a 58% enhancement from using X-ray reflections alone. Furthermore, addition of solid-state NMR restraints greatly improved the overall quality of the structure by promoting 22% of DsbB transmembrane residues into the most favored regions of Ramachandran space in comparison to the crystal structure. This method is widely applicable to any protein system where X-ray data are available, and is particularly useful for the study of weakly diffracting crystals.

Solid-state NMR study of the charge-transfer complex between ubiquinone-8 and disulfide bond generating membrane protein DsbB.

Ubiquinone (Coenzyme Q) plays an important role in the mitochondrial respiratory chain and also acts as an antioxidant in its reduced form, protecting cellular membranes from peroxidation. De novo disulfide bond generation in the E. coli periplasm involves a transient complex consisting of DsbA, DsbB, and ubiquinone (UQ). It is hypothesized that a charge-transfer complex intermediate is formed between the UQ ring and the DsbB-C44 thiolate during the reoxidation of DsbA, which gives a distinctive ~500 nm absorbance band. No enzymological precedent exists for an UQ-protein thiolate charge-transfer complex, and definitive evidence of this unique reaction pathway for DsbB has not been fully demonstrated. In order to study the UQ-8-DsbB complex in the presence of native lipids, we have prepared isotopically labeled samples of precipitated DsbB (WT and C41S) with endogenous UQ-8 and lipids, and we have applied advanced multidimensional solid-state NMR methods. Double-quantum filter and dipolar dephasing experiments facilitated assignments of UQ isoprenoid chain resonances not previously observed and headgroup sites important for the characterization of the UQ redox states: methyls (~20 ppm), methoxys (~60 ppm), olefin carbons (120-140 ppm), and carbonyls (150-160 ppm). Upon increasing the DsbB(C41S) pH from 5.5 to 8.0, we observed a 10.8 ppm upfield shift for the UQ C1 and C4 carbonyls indicating an increase of electron density on the carbonyls. This observation is consistent with the deprotonation of the DsbB-C44 thiolate at pH 8.0 and provides direct evidence of the charge-transfer complex formation. A similar trend was noted for the UQ chemical shifts of the DsbA(C33S)-DsbB(WT) heterodimer, confirming that the charge-transfer complex is unperturbed by the DsbB(C41S) mutant used to mimic the intermediate state of the disulfide bond generating reaction pathway.