title: Resonance assignment of disordered protein with repetitive and overlapping sequence using combinatorial approach reveals initial structural propensities and local restrictions in the denatured state.
abstract: NMR resonance assignment of intrinsically disordered proteins poses a challenge because of the limited dispersion of amide proton chemical shifts. This becomes even more complex with the increase in the size of the system. Residue specific selective labeling/unlabeling experiments have been used to resolve the overlap, but require multiple sample preparations. Here, we demonstrate an assignment strategy requiring only a single sample of uniformly labeled (13)C,(15)N-protein. We have used a combinatorial approach, involving 3D-HNN, CC(CO)NH and 2D-MUSIC, which allowed us to assign a denatured centromeric protein Cse4 of 229 residues. Further, we show that even the less sensitive experiments, when used in an efficient manner can lead to the complete assignment of a complex system without the use of specialized probes in a relatively short time frame. The assignment of the amino acids discloses the presence of local structural propensities even in the denatured state accompanied by restricted motion in certain regions that provides insights into the early folding events of the protein.
authors: Department of Bioscience and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India.
authors: Nikita
authors: N
authors: Malik
authors: Department of Bioscience and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India. askutoshk@iitb.ac.in.
authors: Ashutosh
authors: 0000-0002-5061-8162
authors: A
authors: Kumar
mesh_terms: D000595:Amino Acid Sequence; D064267:Intrinsically Disordered Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011489:Protein Denaturation; D020449:Repetitive Sequences, Amino Acid
chemical_list: D064267:Intrinsically Disordered Proteins
keywords: Centromeric protein; Denatured protein; Intrinsically disordered protein; MUSIC; Protein dynamics