Combined Isothermal Titration and Differential Scanning Calorimetry Define Three-State Thermodynamics of fALS-Associated Mutant Apo SOD1 Dimers and an Increased Population of Folded Monomer

Abstract

Many proteins are naturally homooligomers, homodimers most frequently. The overall stability of oligomeric proteins may be described in terms of the stability of the constituent monomers and the stability of their association; together, these stabilities determine the populations of different monomer and associated species, which generally have different roles in the function or dysfunction of the protein. Here we show how a new combined calorimetry approach, using isothermal titration calorimetry to define monomer association energetics together with differential scanning calorimetry to measure total energetics of oligomer unfolding, can be used to analyze homodimeric unmetalated (apo) superoxide dismutase (SOD1) and determine the effects on the stability of structurally diverse mutations associated with amyotrophic lateral sclerosis (ALS). Despite being located throughout the protein, all mutations studied weaken the dimer interface, while concomitantly either decreasing or increasing the marginal stability of the monomer. Analysis of the populations of dimer, monomer, and unfolded monomer under physiological conditions of temperature, pH, and protein concentration shows that all mutations promote the formation of folded monomers. These findings may help rationalize the key roles proposed for monomer forms of SOD1 in neurotoxic aggregation in ALS, as well as roles for other forms of SOD1. Thus, the results obtained here provide a valuable approach for the quantitative analysis of homooligomeric protein stabilities, which can be used to elucidate the natural and aberrant roles of different forms of these proteins and to improve methods for predicting protein stabilities.

Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)—Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway

“…The increased concentration of dimer could affect the DSC trace by altering the melting process. For the native protein, the unfolding of the dimer is concurrent with the melting of the molten globular C-terminal domain to yield a partially unfolded monomer [51, 52], as observed by the first transition in Figure 3A. Following this concerted melting process, the remainder of the protein melts to yield completely unfolded monomers, represented by the three-state mechanism shown in equation 1.…”

“…The mechanism (equation 2) would now be reflective of a four-state melting process, in which the destabilized, molten globular C-terminal domain of the mutant protein begins to melt (the first transition of three), reducing dimer stability and causing the dimer to unfold in the second melting event (Fig. 2B) [51, 52]. Lastly, the N-terminal domain melts to complete the unfolding process, just as it had for the native.…”

Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy

“…S-S are both predominantly dimeric (42), so that the critical factor in dimer formation is nevertheless posttranslational binding of metal ions and/or disulfide bond formation. Another example illustrating the potential importance of excited states in the SOD1 maturation process, starting from apoSOD1 2SH , is provided by the metal-binding mutants H46R and G85R.…”

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

“…The stability of the SOD1 dimer is affected by ALS-related mutations and several post-translational modifications, which typically bias the SOD1 misfolding pathway toward dimmer dissociation and lead to larger populations of oligomers and aggregates (Broom et al, 2016; Khare et al, 2004; Redler et al, 2014). In familial and sporadic ALS cases, post-translational modifications induced by environmental factors play a significant role in disease progression (Barber and Shaw, 2010; Chattopadhyay et al, 2015; Pasinelli and Brown, 2006).…”