title: Probing the Residual Structure of the Low Populated Denatured State of ADA2h under Folding Conditions by Relaxation Dispersion Nuclear Magnetic Resonance Spectroscopy.
abstract: The structural characterization of low populated states of proteins with accuracy comparable to that achievable for native states is important for understanding the mechanisms of protein folding and function, as well as misfolding and aggregation. Because of the transient nature of these low populated states, they are seldom detected directly under conditions that favor folding. The activation domain of human procarboxypeptidase A2 (ADA2h) is an α/β-protein that forms amyloid fibrils at low pH, presumably initiated from a denatured state with a considerable amount of residual structure. Here we used Carr-Parcell-Meiboom-Gill relaxation dispersion (CPMG RD) nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the denatured state of the ADA2h I71V mutant under conditions that favor folding. Under these conditions, the lifetime of the denatured state of I71V ADA2h is on the order of milliseconds and its population is approximately several percent, which makes this mutant amenable to studies by CPMG RD methods. The nearly complete set of CPMG RD-derived backbone (15)N, (13)C, and (1)H NMR chemical shifts in the I71V ADA2h denatured state reveals that it retains a significant fraction (up to 50-60%) of nativelike α-helical structure, while the regions encompassing native β-strands are structured to a much lesser extent. The nativelike α-helical structure of the denatured state can bring together hydrophobic residues on the same sides of α-helices, making them available for intra- or intermolecular interactions. CPMG RD data analysis thus allowed a detailed structural characterization of the ADA2h denatured state under folding conditions not previously achieved for this protein.
authors.affiliation: †Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut 06030, United States.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Pustovalova
authors.affiliation: ‡Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.
authors.forename: Predrag
authors.initials: P
authors.lastname: Kukic
authors.affiliation: ‡Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.
authors.forename: Michele
authors.initials: M
authors.lastname: Vendruscolo
authors.affiliation: †Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut 06030, United States.
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Korzhnev
mesh_terms: D019943:Amino Acid Substitution; D043422:Carboxypeptidases A / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017433:Protein Structure, Secondary
chemical_list: D043422:Carboxypeptidases A