| Amyotrophic Lateral Sclerosis (ALS) more commonly known as Lou Gehrig's disease, is a malady that characteristically destroys the body's motor neuronal system over a period of a few years. Its pathology characterized by build-up of neuronal protein aggregates, is typical of neurodegenerative diseases. Over 100 mutants of the anti-oxidant enzyme Cu/Zn superoxide dismutase (SOD) have been implicated in a familial form of the disease (FALS), and it has been suggested that the aggregation of these mutants plays a pathological role in the development of the disease. Some of these mutants have been shown to exhibit less affinity for the metal cofactor ions copper and zinc, loss of which has been shown to significantly destabilize the enzyme thermodynamically.; We investigated the kinetic stability and folding efficiency of wild type (WT) and mutants D90A, H46R, L144F, G37R, A4V, G85R SOD, in both their native (holo) and demetallated state (apo), by acid-induced unfolding/folding experiments. Our results show that loss of the cofactor metals is a contributing factor in the kinetic destabilization of the enzyme, with both WT and mutant apo SOD exhibiting unfolding rates 2 orders of magnitude faster than the holo enzyme. This implies a pathway whereby both WT and mutant can become susceptible to aggregation and thus potentially contribute to disease progression. It was also shown that copper plays a dominant role in the kinetic stability of SOD but full kinetic stability cannot be achieved without the presence of the zinc cofactor, therefore loss of either can also predispose the enzyme to aggregate.; Mutations may also destabilize the premature forms of the native enzyme and predispose them to misfold upon folding to the fully native form. Through use of the techniques of stopped-flow fluorescence, right-angle and dynamic light scattering, it was found that L144F SOD displayed the highest refolding efficiency, with over 70% refolding to dimer. G37R and G85R had high tendencies to aggregate, as well as A4V to a lesser extent. The presence of monomeric species in all SOD variants except the WT protein implicates a reduction in refolding efficiency for the mutant and the creation of the aggregation precursor. |
