We combined Spin Noise Tuning Optimum (SNTO) and electric field component-optimized shaped tube to boost sensitivity for NMR-based metabolomics.

An approach for rapid resonance assignments in proteins based on 2D13C-detected NMR experiments combined with amino acid selective unlabeling.

Nuclear magnetic resonance (NMR)-based metabolomics has witnessed rapid advancements in recent years with the continuous development of new methods to enhance the sensitivity, resolution, and speed of data acquisition. Some of the approaches were earlier used for peptide and protein resonance assignments and have now been adapted to metabolomics. At the same time, new NMR methods involving novel data acquisition techniques, suited particularly for high-throughput analysis in metabolomics, have been developed. In this review, we focus on the different sampling strategies or data acquisition methods that have been developed in our laboratory and other groups to acquire NMR spectra rapidly with high sensitivity and resolution for metabolomics. In particular, we focus on the use of multiple receivers, phase modulation NMR spectroscopy, and fast-pulsing methods for identification and assignments of metabolites.

Heme-binding proteins constitute a large family of catalytic and transport proteins. Their widespread presence as globins and as essential oxygen and electron transporters, along with their diverse enzymatic functions, have made them targets for protein design. Most previously reported designs involved the use of α-helical scaffolds, and natural peptides also exhibit a strong preference for these scaffolds. However, the reason for this preference is not well-understood, in part because alternative protein designs, such as those with β-sheets or hairpins, are challenging to perform. Here, we report the computational design and experimental validation of a water-soluble heme-binding peptide, Pincer-1, composed of predominantly β-scaffold secondary structures. Such heme-binding proteins are rarely observed in nature, and by designing such a scaffold, we simultaneously increase the known fold space of heme-binding proteins and expand the limits of computational design methods. For a β-scaffold, two tryptophan zipper β-hairpins sandwiching a heme molecule were linked through an N-terminal cysteine disulfide bond. β-Hairpin orientations and residue selection were performed computationally. Heme binding was confirmed through absorbance experiments and surface plasmon resonance experiments ( = 730 ± 160 nm). CD and NMR experiments validated the β-hairpin topology of the designed peptide. Our results indicate that a helical scaffold is not essential for heme binding and reveal the first designed water-soluble, heme-binding β-hairpin peptide. This peptide could help expand the search for and design space to cytoplasmic heme-binding proteins.