title: Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts.
abstract: The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states. The holo G93A and E100G ALS mutants have moderately decreased global stability, whereas V148I is slightly stabilized. Comparison of the holo mutants as well as the marginally stable immature monomeric unmetalated and disulfide-reduced (apo(2SH)) pWT with holo pWT shows that changes in the local structural stability of individual amides vary greatly, with average changes corresponding to differences in global protein stability measured by differential scanning calorimetry. Mutants also exhibit altered conformational heterogeneity compared to pWT. Strikingly, substantial increases as well as decreases in local stability and conformational heterogeneity occur, in particular upon maturation and for G93A. Thus, the temperature-dependence of amide shifts for SOD1 variants is a rich source of information on the location and extent of perturbation of structure upon covalent changes and ligand binding. The implications for potential mechanisms of toxic misfolding of SOD1 in disease and for general aspects of protein energetics, including entropy-enthalpy compensation, are discussed.
authors: Colleen M
authors: CM
authors: Doyle
authors: Jessica A
authors: JA
authors: Rumfeldt
authors: Helen R
authors: HR
authors: Broom
authors: Ashok
authors: A
authors: Sekhar
authors: Program in Molecular Structure and Function, Hospital for Sick Children , Toronto, Canada.
authors: Lewis E
authors: LE
authors: Kay
authors: Elizabeth M
authors: EM
authors: Meiering
mesh_terms: D000577:Amides / Q000737:chemistry* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning / Q000379:methods; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D013696:Temperature; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000577:Amides; D003300:Copper; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D015032:Zinc