**3. Folding, unfolding and misfolding of SOD1**

A common and powerful approach to understanding the molecular basis for aggregate formation is to investigate the biophysical properties of mutant proteins in the native and unfolded states, as well as any equilibrium or kinetic intermediates that arise as the protein folds or unfolds (Dobson 2004). Equilibrium species refer to the most stable conformations that are significantly populated under specific steady-state conditions, while kinetic species refer to conformations that are transiently populated as an unfolded protein folds into its native conformation. Typically, kinetic folding intermediates have a relatively low energy barrier of formation, and therefore can form quickly, but they are generally not the most stable conformations. Before protein folding has reached equilibrium (ie. during kinetic conditions), it is not the stability of each state that determines the relative population of each species along the folding/unfolding pathway, but rather how rapidly these states can be accessed on the time scale of protein folding/unfolding. Investigating the molecular characteristics that govern the stability of different states and enable efficient folding of SOD1 can provide key insights into the cause of ALS (Rumfeldt et al. 2006; Stathopulos et al. 2006; Vassall et al. 2006; Rumfeldt et al. 2009; Vassall et al. 2011).

In recent years, systematic analyses of the effects of fALS-associated mutations on the stability and folding of various forms of SOD1, including holoS-S, apoS-S and apoSH, have been reported. Human SOD1 contains two free cysteine residues at amino acid positions 6 and 111 (Figure 2), and these free cysteine residues inhibit reversible unfolding of SOD1 *in vitro* by forming intramolecular and intermolecular non-native disulfide bonds, which promote SOD1 aggregation (Lepock et al. 1990; McRee et al. 1990). Reversible unfolding is a prerequisite for thermodynamic analysis, and so to overcome this limitation pseudo-wild type (pWT) constructs lacking these free cysteines have been used extensively for *in vitro* studies of SOD1. In the most widely used pWT construct, the free cysteines are mutated to alanine and serine at positions 6 and 111, respectively (Lepock et al. 1990; McRee et al. 1990; Stathopulos et al. 2003; Rumfeldt et al. 2006; Stathopulos et al. 2006; Vassall et al. 2006; Kayatekin et al. 2008; Rumfeldt et al. 2009; Vassall et al. 2011); however, other mutations at these positions have also been used (most notably C6A and C111A) (Lindberg et al. 2004; Nordlund and Oliveberg 2006; Nordlund et al. 2009). Not only are these chemically and structurally conservative mutations, a serine at position 111 is found in most other mammalian SOD1, and alanine at position 6 is observed in other non-mammalian organisms (Getzoff et al. 1989). Mutating the free cysteines results in highly reversible unfolding of pWT, while having very minimal effects on structure, function and stability (Lepock et al. 1990; McRee et al. 1990; Hallewell et al. 1991; Parge et al. 1992; Vassall et al. 2011). In addition, an engineered monomer construct (pWT*mon* SOD1) has been used to investigate the effects of ALS mutations on the stability and folding behaviour of individual SOD1 subunits (Nordlund and Oliveberg 2006; Hornberg et al. 2007; Kayatekin et al. 2008; Nordlund et al. 2009; Kayatekin et al. 2010). The monomer construct contains two glutamic acid residues in place of Phe50 and Gly51, and the presence of these charged residues in the dimer interface prevents SOD1 dimerization (Bertini et al. 1994; Banci et al. 1998). The use of both pWT and pWT*mon* SOD1 constructs has provided valuable insights into the mechanism of SOD1 folding and misfolding, which are described in the following sections, starting with the most immature to most mature form of SOD1.

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 275

Diverse fALS-associated mutations have markedly different effects on the stability and the folding reversibility of apo2SH SOD1 (Kayatekin et al. 2010; Vassall et al. 2011). Wild-type, pWT, and pWT*mon* apo2SH SOD1 are predominantly folded and can undergo reversible chemical and thermal denaturation, and hence resist aggregation from partially or fully unfolded states (Kayatekin et al. 2010; Vassall et al. 2011). Various fALS-associated mutations decrease the unfolding reversibility, which precludes determination of the free energy of unfolding, and suggests that these mutations increase the aggregation propensity of apo2SH SOD1 (Vassall et al. 2011). Interestingly, an increased tendency to aggregate is not observed for all fALS-associated mutants, in particular not for those with compromised metal binding (Kayatekin et al. 2010; Vassall et al. 2011). Although the free energy of unfolding could not be determined for all mutants studied, owing to the limited stability of this form of the protein, the effects of each mutation on stability could be estimated from their apparent melting temperatures. This revealed that fALS-associated mutations generally have the largest effect on the stability of this most immature form of SOD1 (Table 1), often decreasing the melting temperature of apoSH to below 37°C, and so markedly increasing the fraction of protein that is unfolded at physiological temperature (Furukawa and O'Halloran 2005; Kayatekin et al. 2010; Vassall et al. 2011). Of the mutant SOD1s that unfold reversibly, most show decreased thermodynamic stability, but some (e.g. those involved in metal binding) have little or even a stabilizing effect on stability (Valentine et al. 2005; Kayatekin et al. 2010; Vassall et al. 2011). These findings imply that in some cases the

key effects of mutations in ALS may be manifested in more mature forms of SOD1.

Relatively little is known about the kinetic folding mechanism of apo2SH SOD1. Initial studies of engineered reduced monomer variants have reported 2-state (U ↔ M) folding kinetics, which resembles the behaviour of monomeric apoS-S SOD1s (Lindberg et al. 2004; Kayatekin et al. 2010) (see section 3.4). A study using a monomeric C6A/C111A/C57A/ C146A construct that resembles the apo2SH form, due to its inability to form a disulfide bond and dimerize, showed that disulfide bond formation was not required to facilitate the early contacts made in the monomer folding pathway (Lindberg et al. 2004). Thus, the transition state between unfolded and folded monomers in both the apo2SH and the apoS-S monomer folding pathways may be similar. However, the disulfide bond stabilizes the folded monomer by decreasing the rate of unfolding, thereby increasing the population of

Formation of a disulfide bond between Cys57 and Cys146 greatly diminishes the conformational freedom of Loop IV (Hornberg et al. 2007), and gives rise to energetically favourable dimer formation (Lindberg et al. 2004; Vassall et al. 2006; Ding and Dokholyan 2008; Kayatekin et al. 2010; Vassall et al. 2011). The observed equilibrium folding mechanism of the pWT apoS-S SOD1 dimer depends on the method of inducing denaturation (i.e. chemical denaturant *versus* heat). Spectroscopically-monitored chemical denaturation of pWT apoS-S SOD1 can be described by a 3-state mechanism in which dimer dissociation is followed by monomer unfolding (N2 ↔ 2M ↔ 2U) (Vassall et al. 2006). Due to mass action, however, at increased protein concentrations there is little population of the folded monomer and the mechanism appears 2-state (N2 ↔ 2U) (Lindberg et al. 2004; Svensson et

**3.2 Kinetic unfolding and refolding of apo2SH** 

folded monomer (Lindberg et al. 2004) (see section 3.4).

**3.3 Equilibrium denaturation of apoS-S SOD1** 


Table 1. Stability parameters obtained from DSC measurements of different forms of wild type (WT) and pWT SOD1

a(Stathopulos et al. 2006) The parameters were obtained using average fitted values determined using 0.5 mg mL-1 protein in 20 mM Hepes pH 7.8.

b(Lepock et al. 1990) t*m* defined as the temperature of half completion of the DSC profiles determined using 2-4 mg mL-1 protein in 100 mM phosphate.

<sup>c</sup>G extrapolated to 37°C using methods described in (Stathopulos et al. 2006). Value is in units of per mol dimer.

d(rodriguiz/valentine,2005,PNAS)determined using 2 mg mL-1 protein in 100 mM potassium phosphate pH 7.4.

e(Vassal,2011,PNAS) parameters obtained using average fitted values determined using 0.5 mg mL-1 protein in 20 mM Hepes pH 7.4, 1mM TCEP.

f Value is in units of per mol monomer.

### **3.1 Equilibrium denaturation of apo2SH SOD1**

In its most immature form, with no bound metals and reduced disulfide bond, apo2SH SOD1 adopts a marginally stable folded monomer structure. Chemical and thermal equilibrium denaturation of apo2SH SOD1 is well described by a 2-state unfolding transition between folded and unfolded monomers, denoted as M and U, respectively (M ↔ U) (Kayatekin et al. 2010; Vassall et al. 2011). At 37C and neutral pH, this form of the protein has a low free energy of unfolding, 1.8 kcal mol-1 and 1.6 kcal mol-1 for pWT and WT, respectively (Vassall et al. 2011), indicating that ~95 % and 93 % of the protein is folded. Furthermore, the corresponding melting temperatures are 48 °C and 47 °C, respectively. This stability is relatively low compared to other globular proteins, which typically have unfolding free energies of ~5-15 kcal mol-1 (Jackson 1998) as well as to more mature forms of SOD1 (see sections 3.2-3.4, 3.6, 3.7, Table 1).

Structural investigations by x-ray crystallography and NMR have shown that without the bound metals and disulfide bond, the interface loop (Loop IV) is minimally structured and the dimer interface is disrupted (Arnesano et al. 2004; Hornberg et al. 2007). The NMR solution structure of monomeric (pWT*mon*) with no bound metals but intact disulfide bond, apoS-S (Banci et al. 2003) provides an interesting comparison (Figure 2B). pWT*mon* apoS-S SOD1 adopts an open β-barrel structure due to the flexibility of β-strands 4 and 5, and the inability of the two β-sheets to effectively pack against one another. Furthermore, Loops IV and VII are extensively disordered (Banci et al. 2003; Banci et al. 2010). Disulfide bond reduction promotes further disorder of the marginally stable Loop IV structure by releasing it from its anchor to β-strand 8 (Hornberg et al. 2007). Because Loop IV contains residues required for Zn binding, disulfide bond formation and dimerization, these modifications are thermodynamically coupled. When the disulfide bond is reduced, SOD1 has a much lower affinity for Zn (75 nM *verses* 100 pM for apoS-S SOD1) (Kayatekin et al. 2010), and dimer formation is energetically unfavourable (Arnesano et al. 2004).

**HoloS-S 92.0a (81.7b) 80.1b 33.0a,c ApoS-S 59.0a 52.5d 13.2a,c Apo2SH 47.6e 46.8e 1.8e,f** Table 1. Stability parameters obtained from DSC measurements of different forms of wild

a(Stathopulos et al. 2006) The parameters were obtained using average fitted values

d(rodriguiz/valentine,2005,PNAS)determined using 2 mg mL-1 protein in 100 mM

1998) as well as to more mature forms of SOD1 (see sections 3.2-3.4, 3.6, 3.7, Table 1).

formation is energetically unfavourable (Arnesano et al. 2004).

b(Lepock et al. 1990) t*m* defined as the temperature of half completion of the DSC profiles

<sup>c</sup>G extrapolated to 37°C using methods described in (Stathopulos et al. 2006). Value is in

e(Vassal,2011,PNAS) parameters obtained using average fitted values determined using 0.5

In its most immature form, with no bound metals and reduced disulfide bond, apo2SH SOD1 adopts a marginally stable folded monomer structure. Chemical and thermal equilibrium denaturation of apo2SH SOD1 is well described by a 2-state unfolding transition between folded and unfolded monomers, denoted as M and U, respectively (M ↔ U) (Kayatekin et al. 2010; Vassall et al. 2011). At 37C and neutral pH, this form of the protein has a low free energy of unfolding, 1.8 kcal mol-1 and 1.6 kcal mol-1 for pWT and WT, respectively (Vassall et al. 2011), indicating that ~95 % and 93 % of the protein is folded. Furthermore, the corresponding melting temperatures are 48 °C and 47 °C, respectively. This stability is relatively low compared to other globular proteins, which typically have unfolding free energies of ~5-15 kcal mol-1 (Jackson

Structural investigations by x-ray crystallography and NMR have shown that without the bound metals and disulfide bond, the interface loop (Loop IV) is minimally structured and the dimer interface is disrupted (Arnesano et al. 2004; Hornberg et al. 2007). The NMR solution structure of monomeric (pWT*mon*) with no bound metals but intact disulfide bond, apoS-S (Banci et al. 2003) provides an interesting comparison (Figure 2B). pWT*mon* apoS-S SOD1 adopts an open β-barrel structure due to the flexibility of β-strands 4 and 5, and the inability of the two β-sheets to effectively pack against one another. Furthermore, Loops IV and VII are extensively disordered (Banci et al. 2003; Banci et al. 2010). Disulfide bond reduction promotes further disorder of the marginally stable Loop IV structure by releasing it from its anchor to β-strand 8 (Hornberg et al. 2007). Because Loop IV contains residues required for Zn binding, disulfide bond formation and dimerization, these modifications are thermodynamically coupled. When the disulfide bond is reduced, SOD1 has a much lower affinity for Zn (75 nM *verses* 100 pM for apoS-S SOD1) (Kayatekin et al. 2010), and dimer

**t***m* **WT (°C)** 

*G***, 37°C pWT (kcal mol-1)** 

**SOD1 Form t***m* **pWT** 

type (WT) and pWT SOD1

units of per mol dimer.

f

potassium phosphate pH 7.4.

Value is in units of per mol monomer.

**(°C)**

determined using 0.5 mg mL-1 protein in 20 mM Hepes pH 7.8.

determined using 2-4 mg mL-1 protein in 100 mM phosphate.

mg mL-1 protein in 20 mM Hepes pH 7.4, 1mM TCEP.

**3.1 Equilibrium denaturation of apo2SH SOD1** 

Diverse fALS-associated mutations have markedly different effects on the stability and the folding reversibility of apo2SH SOD1 (Kayatekin et al. 2010; Vassall et al. 2011). Wild-type, pWT, and pWT*mon* apo2SH SOD1 are predominantly folded and can undergo reversible chemical and thermal denaturation, and hence resist aggregation from partially or fully unfolded states (Kayatekin et al. 2010; Vassall et al. 2011). Various fALS-associated mutations decrease the unfolding reversibility, which precludes determination of the free energy of unfolding, and suggests that these mutations increase the aggregation propensity of apo2SH SOD1 (Vassall et al. 2011). Interestingly, an increased tendency to aggregate is not observed for all fALS-associated mutants, in particular not for those with compromised metal binding (Kayatekin et al. 2010; Vassall et al. 2011). Although the free energy of unfolding could not be determined for all mutants studied, owing to the limited stability of this form of the protein, the effects of each mutation on stability could be estimated from their apparent melting temperatures. This revealed that fALS-associated mutations generally have the largest effect on the stability of this most immature form of SOD1 (Table 1), often decreasing the melting temperature of apoSH to below 37°C, and so markedly increasing the fraction of protein that is unfolded at physiological temperature (Furukawa and O'Halloran 2005; Kayatekin et al. 2010; Vassall et al. 2011). Of the mutant SOD1s that unfold reversibly, most show decreased thermodynamic stability, but some (e.g. those involved in metal binding) have little or even a stabilizing effect on stability (Valentine et al. 2005; Kayatekin et al. 2010; Vassall et al. 2011). These findings imply that in some cases the key effects of mutations in ALS may be manifested in more mature forms of SOD1.

### **3.2 Kinetic unfolding and refolding of apo2SH**

Relatively little is known about the kinetic folding mechanism of apo2SH SOD1. Initial studies of engineered reduced monomer variants have reported 2-state (U ↔ M) folding kinetics, which resembles the behaviour of monomeric apoS-S SOD1s (Lindberg et al. 2004; Kayatekin et al. 2010) (see section 3.4). A study using a monomeric C6A/C111A/C57A/ C146A construct that resembles the apo2SH form, due to its inability to form a disulfide bond and dimerize, showed that disulfide bond formation was not required to facilitate the early contacts made in the monomer folding pathway (Lindberg et al. 2004). Thus, the transition state between unfolded and folded monomers in both the apo2SH and the apoS-S monomer folding pathways may be similar. However, the disulfide bond stabilizes the folded monomer by decreasing the rate of unfolding, thereby increasing the population of folded monomer (Lindberg et al. 2004) (see section 3.4).

### **3.3 Equilibrium denaturation of apoS-S SOD1**

Formation of a disulfide bond between Cys57 and Cys146 greatly diminishes the conformational freedom of Loop IV (Hornberg et al. 2007), and gives rise to energetically favourable dimer formation (Lindberg et al. 2004; Vassall et al. 2006; Ding and Dokholyan 2008; Kayatekin et al. 2010; Vassall et al. 2011). The observed equilibrium folding mechanism of the pWT apoS-S SOD1 dimer depends on the method of inducing denaturation (i.e. chemical denaturant *versus* heat). Spectroscopically-monitored chemical denaturation of pWT apoS-S SOD1 can be described by a 3-state mechanism in which dimer dissociation is followed by monomer unfolding (N2 ↔ 2M ↔ 2U) (Vassall et al. 2006). Due to mass action, however, at increased protein concentrations there is little population of the folded monomer and the mechanism appears 2-state (N2 ↔ 2U) (Lindberg et al. 2004; Svensson et

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 277

Therefore, it seems reasonable that many fALS-associated mutations have a large effect on the global stability of apoS-S SOD1. Structural perturbations in one region of the apoS-S SOD1 structure are likely to result in structural perturbations in another region. However, it is clear that fALS-associated mutations have differing effects on the global stability of apoS-S SOD1. In particular, both the apoS-S and apo2SH forms of the metal-binding mutants appear to possess similar stability compared to the wild-type protein (Rodriguez et al. 2005; Vassall et al. 2011). In fact, some fALS-associated mutations increase the stability of apo2SH and apoS-S forms compared to pWT SOD1 (Vassall et al. unpublished data) (Vassall et al. 2011). For example, the mutation H46R increases the stability of both apo2SH and apoS-S SOD1 and the reason for this increase in stability may be due to the introduction of a positively charged side chain into the metal binding pocket of the protein, effectively

The kinetic unfolding mechanism of pWT apoS-S SOD1 can be described by the same 3-state mechanism previously outlined for equilibrium denaturation of the wild-type and pWT apoS-S form, where dimer dissociation is followed by the unfolding of two monomers (Figure 3) (Svensson et al. 2006). The overall observed rate of unfolding depends on four microscopic rate constants: the rate constant for folding (kf), unfolding (ku), monomer association (ka), and dimer dissociation (kd) (Figure 3B); however, under highly denaturing conditions (ie. high denaturant concentration), the marginally stable monomer unfolds rapidly and the rate of the entire unfolding pathway is determined by the rate of dimer dissociation (kd) (Lindberg et al. 2004; Svensson et al. 2006). In contrast, the overall rate of the refolding pathway of wild-type and pWT apoS-S is limited only by the rate of monomer folding. Therefore, once the monomer has folded the rate of monomer association is extremely rapid (Lindberg et al. 2004; Svensson et al. 2006). The transition state for monomer association is similar to the native dimer with respect to the amount of buried surface area (Svensson et al. 2006). This is similar to the transition state between unfolded and folded monomer, which represents the major energetic barrier of the unfolding/refolding pathway. It has been observed that ~70% of the structure that is buried in the monomer intermediate is also buried in the transition state between the unfolded and folded monomer, suggesting that considerable structural rearrangements must occur before that transition state can form (Svensson et al. 2006). Interestingly, under both equilibrium and kinetic conditions, the population of monomer intermediate remains below 0.5% at physiologically relevant protein concentrations, suggesting that the folding pathway of SOD1 has evolved to limit accumulation of marginally stable monomer intermediates (Svensson et al. 2006). Equilibrium denaturation analyses of fALS-associated mutant apoS-S SOD1 demonstrate a decreased stability of both the monomer intermediate and the dimer interface, increasing the population of monomeric intermediate as well as the unfolded monomer (Lindberg et al. 2004; Vassall et al. 2006), which enhances the accessibility of partially folded, high energy

Kinetic unfolding/refolding studies of the monomeric apoS-S SOD1 (pWT*mon*) and the apo2SH forms of SOD1 can be compared to gain an understanding of how disulfide bond formation modulates the SOD1 folding pathway. Both the pWT apo2SH and the pWT*mon* apoS-S forms of SOD1 fold via a 2-state mechanism, whereby the unfolded monomer adopts a folded conformation in one step (U ↔ M) (Lindberg et al. 2004; Nordlund and Oliveberg

mimicking the stabilizing effects of the charged metals.

**3.4 Kinetic unfolding and refolding of apoS-S SOD1** 

states that may give rise to aggregation.

al. 2006; Vassall et al. 2006). Similarly, due to the higher stability of the apoS-S dimer compared to the apoS-S monomer and the high protein concentration requirement, thermal denaturation by differential scanning calorimetry (DSC) of apoS-S pWT SOD1 appears 2 state (N2 ↔ 2U) and so does not provide direct information about the energetics of dimer dissocation (N2 ↔ 2M ) (Stathopulos et al. 2006; Vassall et al. 2006). Thermal denaturation does reveal, however, that the melting temperatures of wild-type and pWT apoS-S SOD1 are approximately 53°C (Rodriguez et al. 2005) and 60°C (Stathopulos et al. 2006), respectively; therefore, the oxidized form of the protein is predominantly folded at physiological temperature. The differences in the reported melting temperatures may be related to different buffer conditions used as well as the folding irreversibility of wild-type SOD1 (Lepock et al. 1992; Chrunyk and Wetzel 1993; Stathopulos et al. 2003). The changes in melting temperatures caused by fALS mutations generally range from -15°C to +2°C; thus, apoS-S SOD1 mutants are also mainly folded at physiological temperature (Rodriguez et al. 2005; Vassall et al. 2006; Kayatekin et al. 2010).

The stability as well as the conformational dynamics of a protein can be assessed using hydrogen-deuterium (H/D) exchange measurements, which can identify regions of the protein undergoing structural opening (Bai et al. 1995). Interestingly, a number of fALSassociated mutants, in particular those that compromise metal binding, display a similar exchange rate as wild type (Rodriguez et al. 2005). However, others increase the rate of structural fluctuations of apoS-S SOD1 (Rodriguez et al. 2005; Prudencio et al. 2009). In particular, some mutants show pronounced opening of the β-barrel around the edge strands at physiological temperatures (Prudencio et al. 2009). Thus, both equilibrium denaturation and H/D exchange experiments reveal that fALS-associated mutations have diverse effects on the stability of apoS-S SOD1, ranging from destabilizing to stabilizing.

Highly reversible chemical denaturation behaviour has enabled accurate measurements of the energetics of both dimer dissociation and monomer unfolding for pWT and mutant apoS-S. Determining how mutations affect the energy, and thus the population, of each species formed along the (un)folding pathway, provides insight into the mechanisms of apoS-S aggregation. Chemical denaturation experiments have revealed that for structurally and chemically diverse mutations, fALS-associated mutations generally decrease the stability of apoS-S SOD1 by destabilizing both the monomer and the dimer interface, with a larger affect on monomer stability compared to the dimer stability (Vassall et al. 2006). Remarkably, the effects of the mutations appear to propagate extensively through the apoS-S form of the protein, inevitably destabilizing the dimer interface (Vassall, K.A. et al. unpublished data) (Khare et al. 2006; Bystrom et al. 2010). Structurally, the apoS-S SOD1 dimer interface is small compared to the amount of solvent exposed surface area (Tainer et al. 1982; Parge et al. 1992); consequently, the dimer interface may be more easily perturbed in the apoS-S state. Moreover, metal loss induces asymmetry in the dynamics of the apoS-S SOD1 monomers, indicative of a structure that is less compact than the holoS-S dimer (Strange et al. 2007). The conformation of both the β-barrel core, in particular β-strand 5, and the functional Loops IV and VII are less rigid in the absence of bound metal (Banci et al. 2009; Teilum et al. 2009). It has been proposed that metal binding is important for shielding the charged residues in Loops IV and VII, which contain too few hydrophobic residues to facilitate close packing with the β-barrel core in the absence of bound metal (Nordlund et al. 2009). Similarly, metal binding may protect against conformational changes in SOD1 that increase hydrophobic exposure (Tiwari et al. 2009).

al. 2006; Vassall et al. 2006). Similarly, due to the higher stability of the apoS-S dimer compared to the apoS-S monomer and the high protein concentration requirement, thermal denaturation by differential scanning calorimetry (DSC) of apoS-S pWT SOD1 appears 2 state (N2 ↔ 2U) and so does not provide direct information about the energetics of dimer dissocation (N2 ↔ 2M ) (Stathopulos et al. 2006; Vassall et al. 2006). Thermal denaturation does reveal, however, that the melting temperatures of wild-type and pWT apoS-S SOD1 are approximately 53°C (Rodriguez et al. 2005) and 60°C (Stathopulos et al. 2006), respectively; therefore, the oxidized form of the protein is predominantly folded at physiological temperature. The differences in the reported melting temperatures may be related to different buffer conditions used as well as the folding irreversibility of wild-type SOD1 (Lepock et al. 1992; Chrunyk and Wetzel 1993; Stathopulos et al. 2003). The changes in melting temperatures caused by fALS mutations generally range from -15°C to +2°C; thus, apoS-S SOD1 mutants are also mainly folded at physiological temperature (Rodriguez et al.

The stability as well as the conformational dynamics of a protein can be assessed using hydrogen-deuterium (H/D) exchange measurements, which can identify regions of the protein undergoing structural opening (Bai et al. 1995). Interestingly, a number of fALSassociated mutants, in particular those that compromise metal binding, display a similar exchange rate as wild type (Rodriguez et al. 2005). However, others increase the rate of structural fluctuations of apoS-S SOD1 (Rodriguez et al. 2005; Prudencio et al. 2009). In particular, some mutants show pronounced opening of the β-barrel around the edge strands at physiological temperatures (Prudencio et al. 2009). Thus, both equilibrium denaturation and H/D exchange experiments reveal that fALS-associated mutations have diverse effects

Highly reversible chemical denaturation behaviour has enabled accurate measurements of the energetics of both dimer dissociation and monomer unfolding for pWT and mutant apoS-S. Determining how mutations affect the energy, and thus the population, of each species formed along the (un)folding pathway, provides insight into the mechanisms of apoS-S aggregation. Chemical denaturation experiments have revealed that for structurally and chemically diverse mutations, fALS-associated mutations generally decrease the stability of apoS-S SOD1 by destabilizing both the monomer and the dimer interface, with a larger affect on monomer stability compared to the dimer stability (Vassall et al. 2006). Remarkably, the effects of the mutations appear to propagate extensively through the apoS-S form of the protein, inevitably destabilizing the dimer interface (Vassall, K.A. et al. unpublished data) (Khare et al. 2006; Bystrom et al. 2010). Structurally, the apoS-S SOD1 dimer interface is small compared to the amount of solvent exposed surface area (Tainer et al. 1982; Parge et al. 1992); consequently, the dimer interface may be more easily perturbed in the apoS-S state. Moreover, metal loss induces asymmetry in the dynamics of the apoS-S SOD1 monomers, indicative of a structure that is less compact than the holoS-S dimer (Strange et al. 2007). The conformation of both the β-barrel core, in particular β-strand 5, and the functional Loops IV and VII are less rigid in the absence of bound metal (Banci et al. 2009; Teilum et al. 2009). It has been proposed that metal binding is important for shielding the charged residues in Loops IV and VII, which contain too few hydrophobic residues to facilitate close packing with the β-barrel core in the absence of bound metal (Nordlund et al. 2009). Similarly, metal binding may protect against conformational changes in SOD1 that

on the stability of apoS-S SOD1, ranging from destabilizing to stabilizing.

2005; Vassall et al. 2006; Kayatekin et al. 2010).

increase hydrophobic exposure (Tiwari et al. 2009).

Therefore, it seems reasonable that many fALS-associated mutations have a large effect on the global stability of apoS-S SOD1. Structural perturbations in one region of the apoS-S SOD1 structure are likely to result in structural perturbations in another region. However, it is clear that fALS-associated mutations have differing effects on the global stability of apoS-S SOD1. In particular, both the apoS-S and apo2SH forms of the metal-binding mutants appear to possess similar stability compared to the wild-type protein (Rodriguez et al. 2005; Vassall et al. 2011). In fact, some fALS-associated mutations increase the stability of apo2SH and apoS-S forms compared to pWT SOD1 (Vassall et al. unpublished data) (Vassall et al. 2011). For example, the mutation H46R increases the stability of both apo2SH and apoS-S SOD1 and the reason for this increase in stability may be due to the introduction of a positively charged side chain into the metal binding pocket of the protein, effectively mimicking the stabilizing effects of the charged metals.

### **3.4 Kinetic unfolding and refolding of apoS-S SOD1**

The kinetic unfolding mechanism of pWT apoS-S SOD1 can be described by the same 3-state mechanism previously outlined for equilibrium denaturation of the wild-type and pWT apoS-S form, where dimer dissociation is followed by the unfolding of two monomers (Figure 3) (Svensson et al. 2006). The overall observed rate of unfolding depends on four microscopic rate constants: the rate constant for folding (kf), unfolding (ku), monomer association (ka), and dimer dissociation (kd) (Figure 3B); however, under highly denaturing conditions (ie. high denaturant concentration), the marginally stable monomer unfolds rapidly and the rate of the entire unfolding pathway is determined by the rate of dimer dissociation (kd) (Lindberg et al. 2004; Svensson et al. 2006). In contrast, the overall rate of the refolding pathway of wild-type and pWT apoS-S is limited only by the rate of monomer folding. Therefore, once the monomer has folded the rate of monomer association is extremely rapid (Lindberg et al. 2004; Svensson et al. 2006). The transition state for monomer association is similar to the native dimer with respect to the amount of buried surface area (Svensson et al. 2006). This is similar to the transition state between unfolded and folded monomer, which represents the major energetic barrier of the unfolding/refolding pathway. It has been observed that ~70% of the structure that is buried in the monomer intermediate is also buried in the transition state between the unfolded and folded monomer, suggesting that considerable structural rearrangements must occur before that transition state can form (Svensson et al. 2006). Interestingly, under both equilibrium and kinetic conditions, the population of monomer intermediate remains below 0.5% at physiologically relevant protein concentrations, suggesting that the folding pathway of SOD1 has evolved to limit accumulation of marginally stable monomer intermediates (Svensson et al. 2006). Equilibrium denaturation analyses of fALS-associated mutant apoS-S SOD1 demonstrate a decreased stability of both the monomer intermediate and the dimer interface, increasing the population of monomeric intermediate as well as the unfolded monomer (Lindberg et al. 2004; Vassall et al. 2006), which enhances the accessibility of partially folded, high energy states that may give rise to aggregation.

Kinetic unfolding/refolding studies of the monomeric apoS-S SOD1 (pWT*mon*) and the apo2SH forms of SOD1 can be compared to gain an understanding of how disulfide bond formation modulates the SOD1 folding pathway. Both the pWT apo2SH and the pWT*mon* apoS-S forms of SOD1 fold via a 2-state mechanism, whereby the unfolded monomer adopts a folded conformation in one step (U ↔ M) (Lindberg et al. 2004; Nordlund and Oliveberg

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 279

et al. 2009; Kayatekin et al. 2010; Vassall et al. 2011). Despite this recent focus, the form of SOD1 that is most relevant to ALS pathogenesis remains unknown. It is clear that some fALS-associated mutations minimally affect the stability and folding kinetics of apo2SH and apoS-S SOD1, which suggests that it is necessary to look beyond the immature forms of SOD

Similar to the equilibrium denaturation pathway of apoS-S SOD1, the observed equilibrium denaturation mechanism of holoS-S SOD1 depends on the mode of denaturation and protein concentration. For pWT SOD1, holoS-S equilibrium denaturation curves fit a reversible 3-state model in which the folded holo native dimer transitions to the unfolded monomer through a folded, metallated, monomeric intermediate (Rumfeldt et al. 2006). The presence of bound metal stabilizes the monomer intermediate far more than the dimer interface. Thus, the dimer is only slightly stronger in holoS-S compared to apoS-S, while the free energy of monomer folding is much higher for holoS-S SOD1 compared to apoS-S SOD1 (Rumfeldt et al. 2006; Vassall et al. 2006). At high protein concentrations, the population of the monomer intermediate is significantly reduced and the equilibrium denaturation mechanism approaches 2-state (Rumfeldt et al. 2006). Similarly, in thermal denaturation by DSC, the monomeric intermediate is not significantly populated and the unfolding appears 2-state (Stathopulos et al. 2006). In both chemical and thermal denaturation of holoS-S SOD1, metals remain bound throughout the transition, although binding is weakened in the monomer intermediate and unfolded state compared to the dimer (Rumfeldt et al. 2006; Stathopulos et al. 2006; Kayatekin et al. 2008). Equilibrium denaturation of holoS-S SOD1 reveals that metal binding significantly increases the free energy of unfolding (Table 1). Consistent with this, the melting temperature of holoS-S SOD1 is ~30°C higher than apoS-S SOD1. Thus, it is likely that the stabilizing effects of metallation as well as disulfide bond formation are intrinsic inhibitors of SOD1 aggregation. Mutations therefore may exert toxicity to the cell by inhibiting SOD1 maturation and/or by promoting metal loss, dimer dissociation and/or disulfide reduction (Tiwari and Hayward 2003; Lindberg et al. 2004; Furukawa and O'Halloran 2005; Furukawa and O'Halloran 2006; Banci et al. 2007; Hornberg

et al. 2007; Ding and Dokholyan 2008; Furukawa et al. 2008; Tiwari et al. 2009).

Comparable to apoS-S SOD1, the equilibrium denaturation mechanism of fALS-associated mutant holoS-S SOD1 remains the same as pWT. In a number of cases mutations have been shown to decrease the stability of holoS-S, by decreasing the stability of the monomer, with less effect on dimerization (Rumfeldt et al. 2006; Stathopulos et al. 2006; Vassall et al. 2006). This decreased stability of the holoS-S monomer can often be attributed to weakened metal binding (Hayward et al. 2002), as metal dissociation results in an increased population of the less stable apo state. Thus, in most cases mutations appear to have a more local effect on the stability of holoS-S SOD1 compared to apoS-S SOD1 (Rumfeldt et al. 2006; Vassall et al. 2006); yet, structural perturbations due to mutation may propagate further if metal binding is compromised since loop dynamics and interface stability are greatly affected by the presence of metals (Valentine et al. 2005; Smith et al. 2007; Museth et al. 2009). The overwhelming majority of fALS-associated mutations destabilize the holo state, but because of its extremely high thermodynamic stability the absolute increase in the amount of unfolded species will still be very small, and thus unlikely to affect aggregation. What seems more likely to impact disease is increased local structural fluctuations that can arise from

to uncover the cause of ALS.

**3.6 Equilibrium denaturation of holoS-S SOD1** 

2006; Kayatekin et al. 2010). Owing to the similar folding/unfolding mechanism of apo2SH and pWT*mon* apoS-S, disulfide bond formation is probably not requisite in the early monomer folding events. Consistent with this notion, the topology of the β-barrel brings Cys57 and Cys146 close in space, promoting disulfide bond formation. Although reduction of the disulfide bond does not prevent formation of the necessary contacts that facilitate monomer folding, the maintenance of the disulfide bond modulates the stability of the SOD1 monomer by decreasing the rate of unfolding and to a smaller extent increasing the rate of folding (Lindberg et al. 2004; Kayatekin et al. 2010).

Removal of Loops IV and VII has little effect on the structure and dynamics of the core apoS-S β-barrel (Nordlund et al. 2009). Additionally, the folding behaviour of pWT*mon* apoS-S SOD1 in the absence of these functional loops remains 2-state; however, this SOD1 construct has a 10-fold increase in the rate of folding, while the rate of unfolding is less affected (Nordlund et al. 2009). Removing the Zn-binding site has little effect on the rate of folding, but decreases the rate of unfolding, stabilizing the folded apoS-S SOD1 monomer (Nordlund et al. 2009). These results suggest that while the β-barrel can fold independently of Loop IV and VII, these critical functional loops endow the protein with a less than optimal folding mechanism and may increase the aggregation propensity of the immature forms of the protein.

The overall rate of both apo2SH and apoS-S SOD1 folding appears to be dictated largely by the structural determinants of the monomer folding nucleus. Furthermore, regions extraneous to this folding nucleus are the more labile regions of the protein that unfold first, and thus have been suggested to play a role in SOD1 aggregation. It has been shown that βstrands 1-3 of the first β-sheet must make contact with β-strands 4 and 7 in the second βsheet to overcome the monomer folding energy barrier. The other strands (β5, β6 and β8) remain disordered in the transition state between unfolded and folded monomers (Nordlund and Oliveberg 2006). By attaching Loop IV to β-strand 8 and forming the dimer interface the disulfide bond may prevent the structural fluctuations that lead to the unravelling of the more dynamic strands of the β-barrel.

### **3.5 Are the metal free forms of SOD1 the common denominator in fALS toxicity?**

Taken together, equilibrium and kinetic folding studies of apo2SH and apoS-S SOD1 suggest that a number of factors may contribute to an increased tendency to aggregate. Many fALS-associated mutations increase the fraction of partially folded monomers, while a few mutations remain wild-type-like in their folding behaviour and aggregation propensity. Because the β-strands display differences in conformational freedom, the location of each fALS-associated mutation and the physicochemical properties of the amino acid introduced seem to greatly affect SOD1 stability and accessibility to partially folded species that may bridge the gap between productive folding and aggregation pathways. These differences may also change the structural properties of the aggregates formed. In recent years, many studies have focused on characterizing a common underlying cause of toxicity in all SOD1 associated fALS cases. Thus a great deal of attention has been directed towards studying the biophysical properties of the more immature forms of SOD1, since fALS-associated mutations have a greater effect on the stability of these forms compared to the fully mature holoS-S form (Lindberg et al. 2004; Furukawa and O'Halloran 2005; Furukawa and O'Halloran 2006; Khare et al. 2006; Nordlund and Oliveberg 2006; Svensson et al. 2006; Smith et al. 2007; Furukawa et al. 2008; Nordlund et al. 2009; Oztug Durer et al. 2009; Tiwari

2006; Kayatekin et al. 2010). Owing to the similar folding/unfolding mechanism of apo2SH and pWT*mon* apoS-S, disulfide bond formation is probably not requisite in the early monomer folding events. Consistent with this notion, the topology of the β-barrel brings Cys57 and Cys146 close in space, promoting disulfide bond formation. Although reduction of the disulfide bond does not prevent formation of the necessary contacts that facilitate monomer folding, the maintenance of the disulfide bond modulates the stability of the SOD1 monomer by decreasing the rate of unfolding and to a smaller extent increasing the

Removal of Loops IV and VII has little effect on the structure and dynamics of the core apoS-S β-barrel (Nordlund et al. 2009). Additionally, the folding behaviour of pWT*mon* apoS-S SOD1 in the absence of these functional loops remains 2-state; however, this SOD1 construct has a 10-fold increase in the rate of folding, while the rate of unfolding is less affected (Nordlund et al. 2009). Removing the Zn-binding site has little effect on the rate of folding, but decreases the rate of unfolding, stabilizing the folded apoS-S SOD1 monomer (Nordlund et al. 2009). These results suggest that while the β-barrel can fold independently of Loop IV and VII, these critical functional loops endow the protein with a less than optimal folding mechanism and may increase the aggregation propensity of the immature

The overall rate of both apo2SH and apoS-S SOD1 folding appears to be dictated largely by the structural determinants of the monomer folding nucleus. Furthermore, regions extraneous to this folding nucleus are the more labile regions of the protein that unfold first, and thus have been suggested to play a role in SOD1 aggregation. It has been shown that βstrands 1-3 of the first β-sheet must make contact with β-strands 4 and 7 in the second βsheet to overcome the monomer folding energy barrier. The other strands (β5, β6 and β8) remain disordered in the transition state between unfolded and folded monomers (Nordlund and Oliveberg 2006). By attaching Loop IV to β-strand 8 and forming the dimer interface the disulfide bond may prevent the structural fluctuations that lead to the

**3.5 Are the metal free forms of SOD1 the common denominator in fALS toxicity?**  Taken together, equilibrium and kinetic folding studies of apo2SH and apoS-S SOD1 suggest that a number of factors may contribute to an increased tendency to aggregate. Many fALS-associated mutations increase the fraction of partially folded monomers, while a few mutations remain wild-type-like in their folding behaviour and aggregation propensity. Because the β-strands display differences in conformational freedom, the location of each fALS-associated mutation and the physicochemical properties of the amino acid introduced seem to greatly affect SOD1 stability and accessibility to partially folded species that may bridge the gap between productive folding and aggregation pathways. These differences may also change the structural properties of the aggregates formed. In recent years, many studies have focused on characterizing a common underlying cause of toxicity in all SOD1 associated fALS cases. Thus a great deal of attention has been directed towards studying the biophysical properties of the more immature forms of SOD1, since fALS-associated mutations have a greater effect on the stability of these forms compared to the fully mature holoS-S form (Lindberg et al. 2004; Furukawa and O'Halloran 2005; Furukawa and O'Halloran 2006; Khare et al. 2006; Nordlund and Oliveberg 2006; Svensson et al. 2006; Smith et al. 2007; Furukawa et al. 2008; Nordlund et al. 2009; Oztug Durer et al. 2009; Tiwari

rate of folding (Lindberg et al. 2004; Kayatekin et al. 2010).

unravelling of the more dynamic strands of the β-barrel.

forms of the protein.

et al. 2009; Kayatekin et al. 2010; Vassall et al. 2011). Despite this recent focus, the form of SOD1 that is most relevant to ALS pathogenesis remains unknown. It is clear that some fALS-associated mutations minimally affect the stability and folding kinetics of apo2SH and apoS-S SOD1, which suggests that it is necessary to look beyond the immature forms of SOD to uncover the cause of ALS.

### **3.6 Equilibrium denaturation of holoS-S SOD1**

Similar to the equilibrium denaturation pathway of apoS-S SOD1, the observed equilibrium denaturation mechanism of holoS-S SOD1 depends on the mode of denaturation and protein concentration. For pWT SOD1, holoS-S equilibrium denaturation curves fit a reversible 3-state model in which the folded holo native dimer transitions to the unfolded monomer through a folded, metallated, monomeric intermediate (Rumfeldt et al. 2006). The presence of bound metal stabilizes the monomer intermediate far more than the dimer interface. Thus, the dimer is only slightly stronger in holoS-S compared to apoS-S, while the free energy of monomer folding is much higher for holoS-S SOD1 compared to apoS-S SOD1 (Rumfeldt et al. 2006; Vassall et al. 2006). At high protein concentrations, the population of the monomer intermediate is significantly reduced and the equilibrium denaturation mechanism approaches 2-state (Rumfeldt et al. 2006). Similarly, in thermal denaturation by DSC, the monomeric intermediate is not significantly populated and the unfolding appears 2-state (Stathopulos et al. 2006). In both chemical and thermal denaturation of holoS-S SOD1, metals remain bound throughout the transition, although binding is weakened in the monomer intermediate and unfolded state compared to the dimer (Rumfeldt et al. 2006; Stathopulos et al. 2006; Kayatekin et al. 2008). Equilibrium denaturation of holoS-S SOD1 reveals that metal binding significantly increases the free energy of unfolding (Table 1). Consistent with this, the melting temperature of holoS-S SOD1 is ~30°C higher than apoS-S SOD1. Thus, it is likely that the stabilizing effects of metallation as well as disulfide bond formation are intrinsic inhibitors of SOD1 aggregation. Mutations therefore may exert toxicity to the cell by inhibiting SOD1 maturation and/or by promoting metal loss, dimer dissociation and/or disulfide reduction (Tiwari and Hayward 2003; Lindberg et al. 2004; Furukawa and O'Halloran 2005; Furukawa and O'Halloran 2006; Banci et al. 2007; Hornberg et al. 2007; Ding and Dokholyan 2008; Furukawa et al. 2008; Tiwari et al. 2009).

Comparable to apoS-S SOD1, the equilibrium denaturation mechanism of fALS-associated mutant holoS-S SOD1 remains the same as pWT. In a number of cases mutations have been shown to decrease the stability of holoS-S, by decreasing the stability of the monomer, with less effect on dimerization (Rumfeldt et al. 2006; Stathopulos et al. 2006; Vassall et al. 2006). This decreased stability of the holoS-S monomer can often be attributed to weakened metal binding (Hayward et al. 2002), as metal dissociation results in an increased population of the less stable apo state. Thus, in most cases mutations appear to have a more local effect on the stability of holoS-S SOD1 compared to apoS-S SOD1 (Rumfeldt et al. 2006; Vassall et al. 2006); yet, structural perturbations due to mutation may propagate further if metal binding is compromised since loop dynamics and interface stability are greatly affected by the presence of metals (Valentine et al. 2005; Smith et al. 2007; Museth et al. 2009). The overwhelming majority of fALS-associated mutations destabilize the holo state, but because of its extremely high thermodynamic stability the absolute increase in the amount of unfolded species will still be very small, and thus unlikely to affect aggregation. What seems more likely to impact disease is increased local structural fluctuations that can arise from

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 281

with dissociation constants (Kd) estimated as 10-18 M and 10-14 M (Crow et al. 1997), respectively. In principal, structural differences between the transition state resembling the free energy barrier between the folded and unfolded monomer, compared to the structure of the folded monomer, determine the regions of the protein that, if changed, will have the largest effect on the rate of unfolding. If a particular region is structured in both the monomer and the transition state, then alterations in the stability of that region, either by metal binding or mutation, will affect the free energy of the monomer and its unfolding transition state in a similar way. As a result, the difference in free energy between the folded monomer and transition state and therefore the rate of monomer unfolding will be unaffected. Alternatively, if a particular region of the protein is structured in the folded monomer, but not in the transition state, then changes in the stability of that region will affect the energetics of the folded monomer and transition state differently. In this case the rate of unfolding will be affected. The Cu binding site is formed by residues in β-strands 4 and 7, which are thought to be structured in the transition state, while the Zn binding site is formed mainly by residues in Loop IV and β-strand 5, which are thought to be disordered in the transition state (Nordlund and Oliveberg 2006). Therefore, the rate of unfolding should be affected more by Zn binding than Cu binding and indeed there is some experimental

It has been shown that fALS-associated mutant holoS-S SOD1s increase the unfolding rates of holoS-S SOD1 (Rumfeldt et al. 2009; Ip et al. 2010). Increased unfolding rates and accessibility of either on or off-folding pathway intermediates may increase the accessibility of transient protein species that can initiate aggregation (Dobson 2003; Wang et al. 2008). Furthermore, it has been proposed that fALS-associated mutations, even those far from the metal binding sites, promote increased levels of Cu-deficient intermediates along the holoS-S SOD1 unfolding pathway (Ip et al. 2010). Cu-deficient intermediates are lower in stability

The *in vitro* refolding mechanism of holoS-S SOD1 is more complex than apoS-S SOD1 due to the presence of metal (Figure 3). While the apoS-S form folds via a simple 3-state (2-step) mechanism, with a rate constant that is limited by the rate of monomer folding (see section 3.4), holoS-S refolding occurs through parallel pathways that differ with respect to the fraction of metal bound to the unfolded and transition states (Rumfeldt et al. 2009). However, in the presence of excess Cu and Zn and at high protein concentrations, conditions that favour metal binding to the unfolded state, the refolding kinetics can be described as a simple 2-step process, as each unfolded SOD1 monomer is saturated with metal. Zn coordination in the native binding site stabilizes the monomer intermediate and folded dimer more than the unfolded monomer, accelerating refolding of apoS-S 100 fold (Kayatekin et al. 2008). However, Zn can also bind to non-native sites on the protein (Kayatekin et al. 2008; Nordlund et al. 2009). The Cu-coordinating residues are capable of binding Zn with micromolar affinity in the denatured state after mutation of the native Zn coordinating residues (Nordlund et al. 2009). This non-native coordination of Zn augments the stability of apoS-S SOD1 by increasing the rate of folding and decreasing the rate of unfolding, but forces SOD1 to adopt a non-native conformation. Thus misligation of Zn can cause misfolding and decrease the efficiency of folding (Nordlund et al. 2009). However, with intact Cu and Zn binding sites, Zn may be coordinated by the Cu-binding site first, helping to overcome the main energy barrier of folding, before moving to the Zn-binding site (Nordlund et al. 2009). Other studies have demonstrated that Zn-binding is required to

evidence that suggests this to be the case (Rumfeldt et al. 2009).

and therefore may have a higher tendency to aggregate.

metal loss and/or dimer dissociation, exposing regions of the SOD1 structure that can make favourable contacts with other SOD1 molecules, and thereby give rise to aggregation from native-like states (Elam et al. 2003; Hwang et al. 2010).

### **3.7 Kinetic unfolding and refolding of holoS-S SOD1**

Analyzing the kinetics of holoS-S SOD1 folding and unfolding provides a method for further characterizing the marginally stable intermediates that form along the folding pathway as these are not always detected at equilibrium where only the most stable species are measurably populated. These kinetic intermediates nevertheless may play significant roles in holoS-S SOD1 aggregation. Both pWT and wild-type holoS-S kinetically unfold slowly *in vitro* via a monomeric intermediate species which has somewhat weaker metal binding affinity relative to the native dimer (Rumfeldt et al. 2006; Kayatekin et al. 2008; Mulligan et al. 2008; Rumfeldt et al. 2009). The overall rate of holoS-S unfolding is dependent on the microscopic rate constants that define each equilibrium transition (Figure 3A).

Fig. 3. The kinetic unfolding mechanism of holoS-S and apoS-S SOD1.

In panel A, the kinetic unfolding mechanism of holoS-S SOD1 is shown. The overall rate of unfolding is dependent on the microscopic rate constants kd, ka, ku, kf, and protein and metal concentration, as well as the equilibrium constant for Zn dissociation from the monomeric intermediate. For a more detailed description refer to (Rumfeldt et al. 2009). In panel B, the simpler kinetic unfolding mechanism of apoS-S SOD1 is shown. The overall rate of unfolding depends only on the microscopic rate constants kd, ka, ku, kf and protein concentration.

The observed unfolding kinetics therefore depend on the rate of dimer dissociation, which occurs rapidly, and the rate of monomer unfolding, a much slower process (Lynch et al. 2004; Rumfeldt et al. 2009). Under high protein concentration conditions, or in the presence of excess metals, pWT holoS-S SOD1 remains fully metallated as it unfolds, with metals remaining bound to the unfolded state, while at low protein concentrations, Zn can dissociate from both the monomeric intermediate and unfolded monomer (Rumfeldt et al. 2009). Similar results were obtained in kinetic unfolding studies of wild-type holoS-S SOD1 in the presence of a metal chelator, where dimer dissociation and Zn loss were found to occur simultaneously, followed by a conformational change in the β-barrel that precedes and facilitates Cu loss (Mulligan et al. 2008).

The differences in Cu and Zn binding as holoS-S SOD1 unfolds can be rationalized by considering the structural differences in the Cu and Zn binding sites, as follows. While SOD1 binds both metals with extremely high affinity, Cu binds with higher affinity than Zn,

metal loss and/or dimer dissociation, exposing regions of the SOD1 structure that can make favourable contacts with other SOD1 molecules, and thereby give rise to aggregation from

Analyzing the kinetics of holoS-S SOD1 folding and unfolding provides a method for further characterizing the marginally stable intermediates that form along the folding pathway as these are not always detected at equilibrium where only the most stable species are measurably populated. These kinetic intermediates nevertheless may play significant roles in holoS-S SOD1 aggregation. Both pWT and wild-type holoS-S kinetically unfold slowly *in vitro* via a monomeric intermediate species which has somewhat weaker metal binding affinity relative to the native dimer (Rumfeldt et al. 2006; Kayatekin et al. 2008; Mulligan et al. 2008; Rumfeldt et al. 2009). The overall rate of holoS-S unfolding is dependent on the microscopic rate constants that define each equilibrium

native-like states (Elam et al. 2003; Hwang et al. 2010).

**3.7 Kinetic unfolding and refolding of holoS-S SOD1** 

Fig. 3. The kinetic unfolding mechanism of holoS-S and apoS-S SOD1.

In panel A, the kinetic unfolding mechanism of holoS-S SOD1 is shown. The overall rate of unfolding is dependent on the microscopic rate constants kd, ka, ku, kf, and protein and metal concentration, as well as the equilibrium constant for Zn dissociation from the monomeric intermediate. For a more detailed description refer to (Rumfeldt et al. 2009). In panel B, the simpler kinetic unfolding mechanism of apoS-S SOD1 is shown. The overall rate

of unfolding depends only on the microscopic rate constants kd, ka, ku, kf and protein

The observed unfolding kinetics therefore depend on the rate of dimer dissociation, which occurs rapidly, and the rate of monomer unfolding, a much slower process (Lynch et al. 2004; Rumfeldt et al. 2009). Under high protein concentration conditions, or in the presence of excess metals, pWT holoS-S SOD1 remains fully metallated as it unfolds, with metals remaining bound to the unfolded state, while at low protein concentrations, Zn can dissociate from both the monomeric intermediate and unfolded monomer (Rumfeldt et al. 2009). Similar results were obtained in kinetic unfolding studies of wild-type holoS-S SOD1 in the presence of a metal chelator, where dimer dissociation and Zn loss were found to occur simultaneously, followed by a conformational change in the β-barrel that precedes

The differences in Cu and Zn binding as holoS-S SOD1 unfolds can be rationalized by considering the structural differences in the Cu and Zn binding sites, as follows. While SOD1 binds both metals with extremely high affinity, Cu binds with higher affinity than Zn,

transition (Figure 3A).

concentration.

and facilitates Cu loss (Mulligan et al. 2008).

with dissociation constants (Kd) estimated as 10-18 M and 10-14 M (Crow et al. 1997), respectively. In principal, structural differences between the transition state resembling the free energy barrier between the folded and unfolded monomer, compared to the structure of the folded monomer, determine the regions of the protein that, if changed, will have the largest effect on the rate of unfolding. If a particular region is structured in both the monomer and the transition state, then alterations in the stability of that region, either by metal binding or mutation, will affect the free energy of the monomer and its unfolding transition state in a similar way. As a result, the difference in free energy between the folded monomer and transition state and therefore the rate of monomer unfolding will be unaffected. Alternatively, if a particular region of the protein is structured in the folded monomer, but not in the transition state, then changes in the stability of that region will affect the energetics of the folded monomer and transition state differently. In this case the rate of unfolding will be affected. The Cu binding site is formed by residues in β-strands 4 and 7, which are thought to be structured in the transition state, while the Zn binding site is formed mainly by residues in Loop IV and β-strand 5, which are thought to be disordered in the transition state (Nordlund and Oliveberg 2006). Therefore, the rate of unfolding should be affected more by Zn binding than Cu binding and indeed there is some experimental evidence that suggests this to be the case (Rumfeldt et al. 2009).

It has been shown that fALS-associated mutant holoS-S SOD1s increase the unfolding rates of holoS-S SOD1 (Rumfeldt et al. 2009; Ip et al. 2010). Increased unfolding rates and accessibility of either on or off-folding pathway intermediates may increase the accessibility of transient protein species that can initiate aggregation (Dobson 2003; Wang et al. 2008). Furthermore, it has been proposed that fALS-associated mutations, even those far from the metal binding sites, promote increased levels of Cu-deficient intermediates along the holoS-S SOD1 unfolding pathway (Ip et al. 2010). Cu-deficient intermediates are lower in stability and therefore may have a higher tendency to aggregate.

The *in vitro* refolding mechanism of holoS-S SOD1 is more complex than apoS-S SOD1 due to the presence of metal (Figure 3). While the apoS-S form folds via a simple 3-state (2-step) mechanism, with a rate constant that is limited by the rate of monomer folding (see section 3.4), holoS-S refolding occurs through parallel pathways that differ with respect to the fraction of metal bound to the unfolded and transition states (Rumfeldt et al. 2009). However, in the presence of excess Cu and Zn and at high protein concentrations, conditions that favour metal binding to the unfolded state, the refolding kinetics can be described as a simple 2-step process, as each unfolded SOD1 monomer is saturated with metal. Zn coordination in the native binding site stabilizes the monomer intermediate and folded dimer more than the unfolded monomer, accelerating refolding of apoS-S 100 fold (Kayatekin et al. 2008). However, Zn can also bind to non-native sites on the protein (Kayatekin et al. 2008; Nordlund et al. 2009). The Cu-coordinating residues are capable of binding Zn with micromolar affinity in the denatured state after mutation of the native Zn coordinating residues (Nordlund et al. 2009). This non-native coordination of Zn augments the stability of apoS-S SOD1 by increasing the rate of folding and decreasing the rate of unfolding, but forces SOD1 to adopt a non-native conformation. Thus misligation of Zn can cause misfolding and decrease the efficiency of folding (Nordlund et al. 2009). However, with intact Cu and Zn binding sites, Zn may be coordinated by the Cu-binding site first, helping to overcome the main energy barrier of folding, before moving to the Zn-binding site (Nordlund et al. 2009). Other studies have demonstrated that Zn-binding is required to

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 283

forms of SOD1 *in vivo* (see section 2). Collectively, experimental findings provide support

Aggregation of the marginally stable apoSH form of SOD1 has been suggested in a number of studies to be particularly important in ALS pathogenesis. Studies from multiple groups have reported that wild-type, pWT and fALS-associated mutant apoSH SOD1 are all predisposed to aggregate (Lindberg et al. 2004; Furukawa and O'Halloran 2005; Hornberg et al. 2007; Chattopadhyay et al. 2008; Furukawa et al. 2008; Oztug Durer et al. 2009; Vassall et al. 2011). Additional support for the biological significance of apoSH aggregation is that fALS-associated mutants expressed in insect cells tend to be more metal deficient and disulfide reduced compared to wild type; this finding was proposed to be a consequence of enhanced opening of the mutant SOD1 structures, exposing the disulfide bond to the reducing environment of the cytosol (Tiwari and Hayward 2003). Furthermore, murine models of fALS have revealed the presence of aggregated disulfide reduced SOD1 species (Jonsson et al. 2006; Zetterstrom et al. 2007). Based on what is known about the relationship between protein stability and aggregation propensity (see section 1.2.1), and on the relatively low stability and the expanded, fluctuating structure of apoSH SOD1 (see section 3.1), it appears that aggregation of this form of SOD1 could be significant under cellular conditions (Furukawa et al. 2008). Consistent with this idea, *in vitro* agitation of apo2SH SOD1 results in amyloid fibril formation (Chattopadhyay et al. 2008; Oztug Durer et al. 2009; Furukawa et al. 2010). Although the agitation phenomenon is poorly understood at a molecular level it may favour amyloid fibril formation over pathways to other morphologies. Agitation may enhance aggregation in general by promoting the formation of aggregation-prone species at air-solution or solid-solution interfaces, as well as increase the rate of aggregation by distributing aggregation nuclei more efficiently and causing preformed aggregates to break and create new nucleation sites (Sicorello et al. 2009). Again, it should be noted that since agitation can promote aggregation of many proteins (Rousseau et al. 2008; Mahler et al. 2009; Sicorello et al. 2009), and ALS is not classified as an amyloid disease (Kerman et al. 2010), it is not yet clear how agitation-induced aggregation *in vitro* is related to aggregation in disease.

Intriguing structural variations in apoSH amyloid and non-amyloid aggregates have been reported and may be related to the different disease durations for different SOD1 mutants. Structural diversity has been observed in the amyloid fibrils formed by different SOD1 mutants (Furukawa et al. 2010). In addition, *in vitro* aggregation experiments without agitation showed that different apo2SH SOD1 mutants may form different sized, small (~40 nm - 1000 nm), soluble, non-amyloid aggregates (Vassall et al. 2011). These soluble species may be particularly relevant to fALS toxicity, as oligomeric protein aggregates have been implicated as key neurotoxic species in many other neurodegenerative diseases (Caughey and Lansbury 2003; Soto and Estrada 2008). The variations in structural characteristics for apo2SH SOD1s aggregates may indicate that different mutants favour distinct aggregation pathways, depending on the aggregation-prone conformations that mutants may adopt.

for contributions from many forms of SOD1 to toxic aggregation in ALS.

**4.1 Aggregation of Apo2SH SOD1** 

**4.1.1 Evidence of Apo2SH SOD1 aggregation** 

**4.1.2 Mechanisms of Apo2SH SOD1 aggregation** 

pre-organize the Cu-binding site (Banci et al. 2003). Together these studies show that metal binding significantly modulates the efficiency of the SOD1 folding pathway by stabilizing both the monomer and dimer and decreasing the rate of unfolding. However, non-native metal binding may force the protein to be kinetically trapped in a partially folded, aggregation prone conformation that is more stable than the unfolded state (Nordlund et al. 2009). Together these results show that the kinetic unfolding and refolding mechanism of SOD1 is highly dependent on metal binding.

### **3.8 fALS mutations and modifications have complex effects on the folding and stability of SOD1**

It is evident that fALS-associated mutants have different and complex effects on the stability of SOD1, and these effects depend on the form of SOD1 being studied. For example, mutations that have a large effect on holoS-S SOD1 stability, such as metal binding mutants, tend to have a much smaller effect on the more immature forms of the protein (Valentine et al. 2005). Also, the effects of mutations are more pronounced, but to varying extents for different mutations, with decreasing stability in immature forms of SOD1 (Vassall et al. 2006; Vassall et al. 2011). It is important to understand how both the equilibrium and kinetic folding pathways of all forms of SOD1 are altered by fALS-associated mutations to untangle the complexity of SOD1 aggregation. Certain mutations may have a large effect on the thermodynamic stability of the protein, through weakened metal binding or by decreasing the stability of the dimer interface, while having a smaller effect on the kinetic stability of SOD1. Other mutations may only subtly alter the thermodynamic stability of the native state, exerting their effects by altering the kinetic stability of SOD1 by decreasing the rate of folding, or by increasing the rate of unfolding (Rumfeldt et al. 2006). These effects can increase the equilibrium and/or transient population of folded or unfolded monomeric species that are prone to aggregate (see section 4).

In addition, fALS mutations may alter the susceptibility of SOD1 to post-translational modifications which will tend to decrease protein stability. These include not only enhanced metal loss and disulfide reduction (resulting in higher population of immature species), but also other modifications, in particular ones that are enhanced under oxidizing cellular conditions, which may occur late in disease. Examples include cysteine oxidation (Gruzman et al. 2007; Karch et al. 2009; Bosco et al. 2010), glutathionylation (Proctor et al. 2011), tryptophan oxidation (Elam et al. 2003), and glycation (Meiering 2008). While there is relatively little quantitative data on the effects of these modifications on stability, there is evidence that they can be destabilizing. Such modifications may play different roles at different disease stages; for example, oxidative modifications may become more pronounced as the disease progresses and contribute to rapid disease progression by enhancing formation of toxic aggregates (Karch et al. 2009).
