title: Rapid NMR Assignments of Proteins by Using Optimized Combinatorial Selective Unlabeling.
abstract: A new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling is presented. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific assignments, without requiring a contiguously stretch of amino acid residues to be linked, and is applicable to deuterated proteins. We show that a 2D [(15) N,(1) H] HSQC spectrum with two 2D spectra can result in ∼50 % assignments. The methodology was applied to two proteins: an intrinsically disordered protein (12 kDa) and the 29 kDa (268 residue) α-subunit of Escherichia coli tryptophan synthase, which presents a challenging case with spectral overlaps and missing peaks. The method can augment existing approaches and will be useful for applications such as identifying active-site residues involved in ligand binding, phosphorylation, or protein-protein interactions, even prior to complete resonance assignments.
authors.affiliation: NMR Research Center, Indian Institute of Science, Malleswaram, Bangalore, 560012, India.|IISc Mathematics Initiative, Indian Institute of Science, Malleswaram, Bangalore, 560012, India.
authors.forename: Abhinav
authors.identifier: http://orcid.org/0000-0002-2392-8050
authors.initials: A
authors.lastname: Dubey
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science-Pilani, Hyderabad Campus, Hyderabad, 500078, India.
authors.forename: Rajashekar Varma
authors.initials: RV
authors.lastname: Kadumuri
authors.affiliation: NMR Research Center, Indian Institute of Science, Malleswaram, Bangalore, 560012, India.
authors.forename: Garima
authors.initials: G
authors.lastname: Jaipuria
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science-Pilani, Hyderabad Campus, Hyderabad, 500078, India. vrk@hyderabad.bits-pilani.ac.in.
authors.forename: Ramakrishna
authors.initials: R
authors.lastname: Vadrevu
authors.affiliation: NMR Research Center, Indian Institute of Science, Malleswaram, Bangalore, 560012, India. hsatreya@sif.iisc.ernet.in.
authors.forename: Hanudatta S
authors.initials: HS
authors.lastname: Atreya
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003903:Deuterium / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000737:chemistry; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000032:analysis; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D014367:Tryptophan Synthase / Q000737:chemistry
chemical_list: D000596:Amino Acids; D018971:Insulin-Like Growth Factor Binding Protein 2; D009587:Nitrogen Isotopes; D011506:Proteins; D003903:Deuterium; D014367:Tryptophan Synthase
keywords: NMR spectroscopy; isotope scrambling; proteins; selective unlabeling assignments; sequence specific resonance; triple resonance experiments