title: A novel tri-enzyme system in combination with laser-driven NMR enables efficient nuclear polarization of biomolecules in solution.
abstract: NMR is an extremely powerful, yet insensitive technique. Many available nuclear polarization methods that address sensitivity are not directly applicable to low-concentration biomolecules in liquids and are often too invasive. Photochemically induced dynamic nuclear polarization (photo-CIDNP) is no exception. It needs high-power laser irradiation, which often leads to sample degradation, and photosensitizer reduction. Here, we introduce a novel tri-enzyme system that significantly overcomes the above challenges, rendering photo-CIDNP a practically applicable technique for NMR sensitivity enhancement in solution. The specificity of the nitrate reductase (NR) enzyme is exploited to selectively in situ reoxidize the reduced photo-CIDNP dye FMNH2. At the same time, the oxygen-scavenging ability of glucose oxidase (GO) and catalase (CAT) is synergistically employed to prevent sample photodegradation. The resulting tri-enzyme system (NR-GO-CAT) enables prolonged sensitivity-enhanced data collection in 1D and 2D heteronuclear NMR, leading to the highest photo-CIDNP sensitivity enhancement (48-fold relative to SE-HSQC) achieved to date for amino acids and polypeptides in solution. NR-GO-CAT extends the concentration limit of photo-CIDNP NMR down to the low micromolar range. In addition, sensitivity (relative to the reference SE-HSQC) is found to be inversely proportional to sample concentration, paving the way for the future analysis of even more diluted samples.
authors.affiliation: Department of Chemistry and Biophysics Program, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706-1322, United States.
authors.forename: Jung Ho
authors.initials: JH
authors.lastname: Lee
authors.forename: Silvia
authors.initials: S
authors.lastname: Cavagnero
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D001234:Aspergillus niger / Q000201:enzymology; D002417:Cattle; D005486:Flavin Mononucleotide / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D007834:Lasers; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010782:Photolysis; D011487:Protein Conformation; D012996:Solutions
chemical_list: D006873:Hydroquinones; D010455:Peptides; D012996:Solutions; C540087:flavin mononucleotide hydroquinone; D005486:Flavin Mononucleotide; D010088:Oxidoreductases
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