The Coupling Between Folding, Zinc Binding, and Disulfide Bond Status of Human Cu, Zn Superoxide Dismutase: A Dissertation
Authors
Kayatekin, CanFaculty Advisor
C. Robert Matthews, Ph.D.Academic Program
Biochemistry and Molecular PharmacologyUMass Chan Affiliations
Biochemistry and Molecular PharmacologyDocument Type
Doctoral DissertationPublication Date
2010-06-15Keywords
Superoxide DismutaseAmyotrophic Lateral Sclerosis
Zinc
Protein Folding
Proteostasis Deficiencies
Disulfides
Amino Acids, Peptides, and Proteins
Biochemical Phenomena, Metabolism, and Nutrition
Biochemistry, Biophysics, and Structural Biology
Enzymes and Coenzymes
Genetic Phenomena
Inorganic Chemicals
Nervous System Diseases
Nutritional and Metabolic Diseases
Organic Chemicals
Metadata
Show full item recordAbstract
Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were >50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate.DOI
10.13028/t2xs-2452Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31852Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/t2xs-2452