When combined with 2D Electronic Reference To access In vivo Concentrations (ERETIC) protocols, the protocol allows for the direct extraction of in vivo metabolite concentrations without the use of internal standards that can be detrimental to living organisms. An improved decoupling approach derived using optimal control theory is presented here that improves the accuracy of metabolite concentrations that can be extracted in vivo down to micromolar concentrations. The results show the perfect-HSQC experiment performs very well in vivo, but the decoupling scheme used is critical for accurate quantitation. This study represents a simple pilot study that compares two of the most popular quantitative 2D HSQC approaches to determine if quantitative results can be directly obtained in vivo in isotopically enriched Daphnia magna (water flea). While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D 1H- 13C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. The separation line (also called ‘Yin-Yang’ diameter) formed by two semicircles of the ‘Tai-Chi’ symbol generate more binary interaction than does the linear diameter.In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. The architecture of homodimeric Sso7c4 is presented as the Chinese traditional ‘Tai-Chi’ symbol. These arrangements form a strand-switched dimer interface. In addition, another short β-sheet is constructed by the β2 strand from each monomer. Association of the monomers is through two β-sheets in each sheet, two strands derive from one monomer and the first strand forms the other monomer. The two monomers are colored differently for clarity. ( D) The individual secondary structure elements are indicated in the ribbon diagram. ( C) NMR ensemble of the selected structures is shown with a backbone chain. ( B) Topology diagram of the Sso7c4 structure showing the connectivity between strands in two β-sheets. The amide protons with very slow exchange rates are circled. Long-range NOEs between β-strands are indicated by double arrows. ( A) The proton nuclear Overhauser enhancement (NOE) networks of the swapped six-stranded and the short two-stranded antiparallel β-sheets of Sso7c4 are defined from the NOEs and amide exchange rate. Topology and NMR structure of the dimeric Sso7c4 protein. furiosus TK, Thermococcus kodakaraensis PHS, P. The scale bar indicates the distance corresponding to 0.1 amino acid substitutions per site. The protein names are colored according to the classification as in (A). ( B) The phylogenetic tree for the above alignment. The residues R11 and R22 are highly conserved across the species and other hydrophobic residues are also important in hydrophobic interactions. Residues are numbered according to Sso7c4a from S. For the group I (Sso7c3, Sso7c4, Saci0101, Saci1212 and STS096) and the group 2, each possessing shorter sequence length near 55 and 75 amino acids, respectively, are aligned. Sequences were obtained form the National Center for Biotechnology Information database ( ). The alignment was performed with the T-coffee server (30). ( A) Sequence alignment of the Sso7c4 family from several archaeas with the corresponding secondary structural elements noted. Comparison of Primary Sequences of Sso7c4 protein family in Archaeal kingdom.
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