In this work, the 3-D structure of the well-known opioid drug heroin in a solution was investigated. The goal was to provide a complete and detailed description of the stable conformers with their relative abundances. This knowledge is very important from the pharmaceutical and forensic point of view as it could help significantly with deeper understanding of heroin's metabolism and the development of antagonist medicines for the case of an overdose. As heroin is a chiral compound with five stereogenic centres, the methods of chiroptical spectroscopy supplemented by density functional theory (DFT) calculations were applied to study its conformations in chloroform solution. The selected chiroptical methods, namely, electronic circular dichroism (ECD) and vibrational circular dichroism (VCD), are inherently sensitive to the 3-D structure of small- to medium-sized chiral organic molecules. A thorough conformational analysis revealed four stable conformers of heroin in chloroform solution, where the conductor-like polarizable continuum model of the solvent was used for all the calculations. The simulated ultraviolet (UV), infrared (IR), ECD, and VCD spectra were compared with the experimental ones and very good agreement was found, which enabled a detailed structure description and interpretation of the spectra. Chiroptical spectroscopy in combination with DFT calculations proved to be a very sensitive tool for the analysis of the 3-D structure of heroin in a solution in contrast with conventional spectroscopic methods. Especially, the application of VCD seems to be a promising approach for monitoring structural changes, for instance, those caused by solvents or interactions with other agents.
Structure determination of RNA-protein complexes is essential for our understanding of the multiple layers of RNA-mediated posttranscriptional regulation of gene expression. Over the past 20years, NMR spectroscopy became a key tool for structural studies of RNA-protein interactions. Here, we review the progress being made in NMR structure determination of large ribonucleoprotein assemblies. We discuss approaches for the design of RNA-protein complexes for NMR structural studies, established and emerging isotope and segmental labeling schemes suitable for large RNPs and how to gain distance restraints from NOEs, PREs and EPR and orientational information from RDCs and SAXS/SANS in such systems. The new combination of NMR measurements with MD simulations and its potential will also be discussed. Application and combination of these various methods for structure determination of large RNPs will be illustrated with three large RNA-protein complexes (>40kDa) and other interesting complexes determined in the past six and a half years.
- MeSH
- Isotope Labeling methods MeSH
- Isotopes chemistry MeSH
- Nucleic Acid Conformation MeSH
- Protein Conformation MeSH
- Models, Molecular MeSH
- Nuclear Magnetic Resonance, Biomolecular methods MeSH
- Proteins chemistry MeSH
- Ribonucleoproteins chemistry MeSH
- RNA chemistry MeSH
- Binding Sites MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Mouse Nkrp1a receptor is a C-type lectin-like receptor expressed on the surface of natural killer cells that play an important role against virally infected and tumor cells. The recently solved crystal structure of Nkrp1a raises questions about a long loop region which was uniquely extended from the central region in the crystal. To understand the functional significance of the loop, the solution structure of Nkrp1a using nuclear magnetic resonance (NMR) spectroscopy was determined. A notable difference between the crystal and NMR structure of Nkrp1a appears in the conformation of the long loop region. While the extended loop points away from the central core and mediates formation of a domain swapped dimer in the crystal, the solution structure is monomeric with the loop tightly anchored to the central region. The findings described the first solution structure in the Nkrp1 family and revealed intriguing similarities and differences to the crystal structure. Proteins 2016; 84:1304-1311. © 2016 Wiley Periodicals, Inc.
- MeSH
- Amino Acid Motifs MeSH
- Escherichia coli genetics metabolism MeSH
- Gene Expression MeSH
- Cloning, Molecular MeSH
- Crystallography, X-Ray MeSH
- NK Cell Lectin-Like Receptor Subfamily B chemistry genetics metabolism MeSH
- Magnetic Resonance Spectroscopy MeSH
- Mice MeSH
- Protein Domains MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Protein Structure, Secondary MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The structure of the active site in a metalloenzyme can be a key determinant of its metal ion binding affinity and catalytic activity. In this study, the conformational features of the Zn(2+)-binding HNH motif were investigated by CD-spectroscopy in combination with isothermal microcalorimetric titrations. Various point mutations, including T454A, K458A and W464A, were introduced into the N-terminal loop of the nuclease domain of colicin E7 (NColE7). We show that the folding of the proteins was severely disturbed by the mutation of the tryptophan residue. This points to the importance of W464, being a part of the hydrophobic core located close to the HNH-motif. ITC demonstrated that the Zn(2+)-binding of the mutants including the W464 site became weak, and according to CD-spectroscopic measurements the addition of the metal ion itself cannot fully recover the functional structure. Titrations with Zn(2+)-ion in the presence and absence of the Im7 protein proved that the structural changes in the unfolded mutant included the HNH-motif itself. The metal-binding of the NColE7 mutants could be, however, fully rescued by the complexation of Im7. This suggests that the formation of a preorganized metal-binding site--existing in the wild-type enzyme but not in the W464 mutants--was induced by Im7. The low nuclease activity of all W464A mutants, however, implies that the interactions of this tryptophan residue are required for precise location of the catalytic residues, i.e. for stabilization of the fine-structure and of the tertiary structure. Our results contribute to the understanding of the metal binding site preorganization.
- MeSH
- Amino Acid Motifs MeSH
- Circular Dichroism MeSH
- Electrophoresis, Agar Gel MeSH
- Endonucleases chemistry MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Calorimetry MeSH
- Models, Molecular MeSH
- Solutions chemistry MeSH
- Protein Structure, Tertiary MeSH
- Binding Sites MeSH
- Zinc chemistry MeSH
- Publication type
- Journal Article MeSH
Hydration of neutral and cationic imidazole is studied by means of ab initio and molecular dynamics calculations, and by photoelectron spectroscopy of the neutral species in a liquid microjet. The calculations show the importance of long range solvent polarization and of the difference between the structure of water molecules in the first shell around the neutral vs cationic species for determining vertical and adiabatic ionization potentials. The vertical ionization potential of neutral imidazole of 8.06 eV calculated using a nonequilibrium polarizable continuum model agrees well with the value of 8.26 eV obtained experimentally for an aqueous solution at pH 10.6.
