Improved strategies for the determination of protein structures from NMR data: The solution structure of acyl carrier protein

AbstractThe hybrid method that combines the early stages of a distance geometry program with simulated annealing in the presence of NMR constraints was optimized to obtain structures fully consistent with the observed NMR data. This was achieved by using more restrictive bounds of the NOE constraints than those usually used in the literature and by grouping the NOEs into classes dependent on the quality of the experimental NOE data. The ‘floating’ stereospecific assignment introduced at the simulated annealing stage of the calculations further improved the definition of the local conformation. An improved sampling and convergence property of the hybrid method was obtained by means of fitting the substructure obtained from the distance geometry program to different conformations. Compared to the standard hybrid methods, this procedure gave superior structures for a 77 amino acid protein, acyl carrier protein from Escherichia coli

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

Solid state NMR/Biophysical Organic Chemistr

A software framework for analysing solid-state MAS NMR data

Solid-state magic-angle-spinning (MAS) NMR of proteins has undergone many rapid methodological developments in recent years, enabling detailed studies of protein structure, function and dynamics. Software development, however, has not kept pace with these advances and data analysis is mostly performed using tools developed for solution NMR which do not directly address solid-state specific issues. Here we present additions to the CcpNmr Analysis software package which enable easier identification of spinning side bands, straightforward analysis of double quantum spectra, automatic consideration of non-uniform labelling schemes, as well as extension of other existing features to the needs of solid-state MAS data. To underpin this, we have updated and extended the CCPN data model and experiment descriptions to include transfer types and nomenclature appropriate for solid-state NMR experiments, as well as a set of experiment prototypes covering the experiments commonly employed by solid-sate MAS protein NMR spectroscopists. This work not only improves solid-state MAS NMR data analysis but provides a platform for anyone who uses the CCPN data model for programming, data transfer, or data archival involving solid-state MAS NMR data

NMR quality control of fragment libraries for screening

Fragment-based screening has evolved as a remarkable approach within the drug discovery process both in the industry and academia. Fragment screening has become a more structure-based approach to inhibitor development, but also towards development of pathway-specific clinical probes. However, it is often witnessed that the availability, immediate and long-term, of a high quality fragment-screening library is still beyond the reach of most academic laboratories. Within iNEXT (Infrastructure for NMR, EM and X-rays for Translational research), a EU-funded Horizon 2020 program, a collection of 782 fragments were assembled utilizing the concept of "poised fragments" with the aim to facilitate downstream synthesis of ligands with high affinity by fragment ligation. Herein, we describe the analytical procedure to assess the quality of this purchased and assembled fragment library by NMR spectroscopy. This quality assessment requires buffer solubility screening, comparison with LC/MS quality control and is supported by state-of-the-art software for high throughput data acquisition and on-the-fly data analysis. Results from the analysis of the library are presented as a prototype of fragment progression through the quality control process

Characterization of pheophytin ground states in Rhodobacter sphaeroides R26 photosynthetic reaction centers from multispin pheophytin enrichment and 2-D 13C MAS NMR dipolar correlation spectroscopy

The electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from C-13 chemical shift data, A new experimental approach involving multispin C-13 labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly C-13 biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the multispin labeled samples 1-D and 2-D solid-state C-13 magic angle spinning NMR spectra could be obtained and used to characterize the pheophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state C-13-C-13 correlation spectra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many C-13 resonances of the [U-C-13]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experiments. Parts of the pheophytins interacting with the protein, at the level of C-13 shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer.Solid state NMR/Biophysical Organic ChemistryBiological and Soft Matter Physic

TapA acts as specific chaperone in TasA filament formation by strand complementation

Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell-cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of β-sandwich subunits. The secondary structure around the intercalated N-terminal strand β0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface

Aspects of receptor binding and signalling of interleukin-4 investigated by site-directed mutagenesis and NMR spectroscopy

Cytokines are hormones that carry information from ceJI to ceH. This information is read from their surface upon binding to transmembrane receptors and by the subsequent initiation of receptor oligomerization. An inftuence on this process through mutagenesis on the hormone surface is highly desirab)e for medical reasons. However, an understanding of hormone-receptor interactions requires insight into the structural changes introduced by the mutations. In this line structural studies on human TL-4 and the medically important IL-4 antagonists YI24D and Y124G are presented. The site a.round YI24 is an important epitope responsible for the a.bility of 11-4 t.o ca.use a signal in the target cells. It is shown that the local main-chain structure around residue 124 in the variants remains unchanged. A strategy is presented here which allows the study of these types of proteins and their variants by NMR which does not require carbon Iabeiied sa.mples