Institutes
Refine
Document Type
- Article (6) (remove)
Language
- English (6)
Has Fulltext
- yes (6)
Is part of the Bibliography
- no (6)
Keywords
- NMR (6) (remove)
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.
tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.
Ribonucleic acid oligonucleotides (RNAs) play pivotal roles in cellular function (riboswitches), chemical biology applications (SELEX-derived aptamers), cell biology and biomedical applications (transcriptomics). Furthermore, a growing number of RNA forms (long non-coding RNAs, circular RNAs) but also RNA modifications are identified, showing the ever increasing functional diversity of RNAs. To describe and understand this functional diversity, structural studies of RNA are increasingly important. However, they are often more challenging than protein structural studies as RNAs are substantially more dynamic and their function is often linked to their structural transitions between alternative conformations. NMR is a prime technique to characterize these structural dynamics with atomic resolution. To extend the NMR size limitation and to characterize large RNAs and their complexes above 200 nucleotides, new NMR techniques have been developed. This Minireview reports on the development of NMR methods that utilize detection on low-γ nuclei (heteronuclei like 13C or 15N with lower gyromagnetic ratio than 1H) to obtain unique structural and dynamic information for large RNA molecules in solution. Experiments involve through-bond correlations of nucleobases and the phosphodiester backbone of RNA for chemical shift assignment and make information on hydrogen bonding uniquely accessible. Previously unobservable NMR resonances of amino groups in RNA nucleobases are now detected in experiments involving conformational exchange-resistant double-quantum 1H coherences, detected by 13C NMR spectroscopy. Furthermore, 13C and 15N chemical shifts provide valuable information on conformations. All the covered aspects point to the advantages of low-γ nuclei detection experiments in RNA.
An automated NMR chemical shift assignment algorithm was developed using multi-objective optimization techniques. The problem is modeled as a combinatorial optimization problem and its objective parameters are defined separately in different score functions. Some of the heuristic approaches of evolutionary optimization are employed in this problem model. Both, a conventional genetic algorithm and multi-objective methods, i.e., the non-dominated sorting genetic algorithms II and III (NSGA2 and NSGA3), are applied to the problem. The multi-objective approaches consider each objective parameter separately, whereas the genetic algorithm followed a conventional way, where all objectives are combined in one score function. Several improvement steps and repetitions on these algorithms are performed and their combinations are also created as a hyper-heuristic approach to the problem. Additionally, a hill-climbing algorithm is also applied after the evolutionary algorithm steps. The algorithms are tested on several different datasets with a set of 11 commonly used spectra. The test results showed that our algorithm could assign both sidechain and backbone atoms fully automatically without any manual interactions. Our approaches could provide around a 65% success rate and could assign some of the atoms that could not be assigned by other methods.
NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.
RcsF, a proposed auxiliary regulator of the regulation of capsule synthesis (rcs) phosphorelay system, is a key element for understanding the RcsC-D-A/B signaling cascade, which is responsible for the regulation of more than 100 genes and is involved in cell division, motility, biofilm formation, and virulence. The RcsC-D-A/B system is one of the most complex bacterial signal transduction pathways, consisting of several membrane-bound and soluble proteins. RcsF is a lipoprotein attached to the outer membrane and plays an important role in activating the RcsC-d-A/B pathway. The exact mechanism of activation of the rcs phosphorelay by RcsF, however, remains unknown. We have analyzed the sequence of RcsF and identified three structural elements: 1) an N-terminal membrane-anchored helix (residues 3-13), 2) a loop (residues 14-48), and 3) a C-terminal folded domain (residues 49-134). We have determined the structure of this C-terminal domain and started to investigate its interaction with potential partners. Important features of its structure are two disulfide bridges between Cys-74 and Cys-118 and between Cys-109 and Cys-124. To evaluate the importance of this RcsF disulfide bridge network in vivo, we have examined the ability of the full-length protein and of specific Cys mutants to initiate the rcs signaling cascade. The results indicate that the Cys-74/Cys-118 and the Cys-109/Cys-124 residues correlate pairwise with the activity of RcsF. Interaction studies showed a weak interaction with an RNA hairpin. However, no interaction could be detected with reagents that are believed to activate the rcs phosphorelay, such as lysozyme, glucose, or Zn(2+) ions.