Structure of proteins in eukaryotic compartments. Pruning the ALS-associated protein SOD1 for in-cell NMR. Investigation of quadruplex structure under physiological conditions using in-cell NMR. Hänsel, R., Foldynová-Trantírková, S., Dötsch, V. Protein nuclear magnetic resonance under physiological conditions. NMR observation of Tau in Xenopus oocytes. In situ observation of protein phosphorylation by high-resolution NMR spectroscopy. Structural disorder of monomeric α-synuclein persists in mammalian cells. Observation of NMR signals from proteins introduced into living mammalian cells by reversible membrane permeabilization using a pore-forming toxin, streptolysin O. Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes. Selenko, P., Serber, Z., Gadea, B., Ruderman, J. Quinary structure modulates protein stability in cells. Monteith, W.B., Cohen, R.D., Smith, A.E., Guzman-Cisneros, E. Probing protein quinary interactions by in-cell nuclear magnetic resonance spectroscopy. Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Residue level quantification of protein stability in living cells. In-cell NMR characterization of the secondary structure populations of a disordered conformation of α-synuclein within E. Protein structure determination in living cells by in-cell NMR spectroscopy. Atomic-resolution monitoring of protein maturation in live human cells by NMR. High-resolution multi-dimensional NMR spectroscopy of proteins in human cells. Looking into live cells with in-cell NMR spectroscopy. The entire procedure takes 4 d from cell culture seeding to NMR data collection. Uniform 15N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. 1H and 1H– 15N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2–3 d, after which the NMR sample is prepared. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. On Line Database of Ensemble Representatives And DOmains (OLDERADO) provides analysis of clustering and domain composition for NMR structure ensembles.In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. The whole ensemble is deposited in the PDB. The outcome of MD simulation is an ensemble of structures (usually 10-20) which, when combined, best satisfy the experimental data. These short distances constitute constraints for molecular dynamics (MD) simulation software, which attempt to satisfy as many of them as possible. The measurements in NMR spectroscopy are a number of different complex spectra that report, among other things, on the chemical environment for the magnetically active nuclei (most commonly 1H, 13C and 15N), on chemical bond connections between nuclei, and on short distances between specific atoms. Typical data collection may take 2-3 weeks for a small soluble protein, but can be substantially longer for larger systems. It utilises the fact that some atomic nuclei are magnetically active and can emit radio frequency signals when placed in a strong external magnetic field (on the order of 10-20 Tesla, which is almost a million times stronger than the Earth’s magnetic field on the surface). Nuclear magnetic resonance (NMR) spectroscopy (for the time being) is the second most common method of structure determination, providing ~7% of all entries in the PDB.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |