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J. Biol. Chem., Vol. 281, Issue 10, 6184-6193, March 10, 2006

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Calorimetric Analysis of Thermodynamic Stability and Aggregation for Apo and Holo Amyotrophic Lateral Sclerosis-associated Gly-93 Mutants of Superoxide Dismutase^*

Peter B. Stathopulos, Jessica A. O. Rumfeldt, Farhad Karbassi, Clare A. Siddall, James R. Lepock, and Elizabeth M. Meiering¹

From the Departments of Chemistry and Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 and the Department of Medical Biophysics and the Ontario Cancer Institute, University of Toronto, Toronto, Ontario M5G 2M9, Canada

Differential scanning calorimetry was used to measure changes in thermodynamic stability and aggregation for glycine 93 mutants of human copper, zinc-superoxide dismutase (SOD). Glycine 93 is a conserved residue at position i + 3 of a tight turn and has been found to be a mutational hot spot in familial amyotrophic lateral sclerosis (fALS). The fALS-associated mutations, G93A, G93S, G93R, G93D, and G93V, were made in a pseudo wild-type background containing no free cysteines, which prevented the formation of aberrant disulfide bonds upon thermal unfolding, and enabled quantitative thermodynamic analysis of the effects of the mutations. Thermal unfolding was highly reversible for all the SODs in both the fully metallated (holo) and metal-free (apo) forms. The data for all the holo-SODs and for the apo-pseudo-wild-type SOD were well fit by a 2-state unfolding model for native dimer (N₂) to two unfolded monomers (2U), N₂ 2U. The holo- and apo-forms of the mutants are significantly destabilized (by 1.5–3.5 kcal mol^–1 monomer) relative to the corresponding forms of pseudo wild-type, with the relative stabilities being correlated with statistical preferences for amino acids in this structural context. Although van't Hoff (H_vH) to calorimetric (H_cal) enthalpy ratios are close to unity for all the holo-SODs and for apo-pseudo-wild-type, consistent with a 2-state transition, H_vH is considerably larger than H_cal for all the apo-mutants. This suggests that the mutations cause apo-SOD to have an increased propensity to misfold or aggregate, which may be linked to increased toxic mutant SOD aggregation in fALS.

Received for publication, August 29, 2005 , and in revised form, December 13, 2005.

^* This work was supported by the Neuromuscular Research Partnership, an initiative of the Amyotrophic Lateral Sclerosis Society of Canada, Muscular Dystrophy Canada, and Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1–S3 and Table SI.

In memory of James R. Lepock, our mentor, colleague, and friend.

¹ To whom correspondence should be addressed: Dept. of Chemistry, University of Waterloo, 200 University Ave., Waterloo, Ontario N2L 3G1, Canada. Tel.: 519-885-1211 (ext. 2254); Fax: 519-746-0435; E-mail: meiering{at}uwaterloo.ca.

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