**1. Introduction**

264 Amyotrophic Lateral Sclerosis

Vonk, W.I.M., Wijmenga, C., Berger, R., van de Sluis, B. & Klomp, L.W.J. (2010). Cu,Zn

Zelko, I.N., Mariani, T.J. & Folz, R.J. (2002). Superoxide dismutase multigene family: a

*Journal of Biological Chemistry*, Vol.285, No.37, (2010), pp. 28991 -29000. Xu, Z., Jung, C., Higgins, C., Levine, J. & Kong, J. (2004). Mitochondrial degeneration in

395-399.

337-49.

Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1.

amyotrophic lateral sclerosis. *J Bioenerg Biomembr*, Vol.36, No.4, (August 2004), pp.

comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. *Free Radic Biol Med*, Vol.33, No.3, (2002), pp.

### **1.1 ALS and SOD1**

In 1993, a genetic link was established between amyotrophic lateral sclerosis (ALS) and mutant forms of Cu,Zn superoxide dismutase (SOD1) (Deng et al. 1993; Rosen et al. 1993), an antioxidant enzyme that catalyzes the dismutation of the damaging free radical superoxide anion (O2-) to hydrogen peroxide (H2O2) and diatomic oxygen (O2) via cyclic reduction and oxidation of a protein-bound Cu ion (Valentine et al. 2005). Today, over 150, predominantly missense mutations have been identified at ~75 sites spread throughout the protein (http://alsod.iop.kcl.ac.uk/). SOD1 mutations are found in ~15- 20% of inherited or familial ALS (fALS) cases and in a small percentage of sporadic ALS (sALS) cases (Rosen et al. 1993; Kato et al. 2000; Liu et al. 2009; Forsberg et al. 2011). fALS accounts for ~10% of all ALS cases and so SOD1 mutations comprise ~1.5-2% of all ALS cases, but nevertheless represent a major known cause of the disease. The clinical symptoms of fALS and sALS are similar, yet fALS patients with SOD1 mutations have an earlier age of disease onset than sALS (by ~10 years) (Wijesekera and Leigh 2009). Furthermore, while the age of disease onset has not been identified as statistically different between different SOD1 mutations, disease duration for each mutation is often different, ranging from shorter (e.g. ~1 year for A4V, the most common mutation in North America) than the typical 3-5 years to longer (e.g. ~18 years for H46R) (Cudkowicz et al. 1997; Valentine et al. 2005; Wang et al. 2008). In humans and murine models of ALS, mutations in the gene encoding SOD1 are typically autosomal dominant and are associated with a toxic gain of function. Despite extensive research, the molecular basis for mutant SOD1 toxicity remains unclear (Valentine et al. 2005; Boillee et al. 2006; Ilieva et al. 2009). Extensive research has been conducted on SOD1-linked fALS, as understanding and treatment of this disease may be relevant to ALS in general. While ALS patients share many clinical symptoms, numerous genes have been linked to ALS, and there is evidence for differences in pathology related to both genetic and environmental factors; hence, ALS is a syndrome and not a single disease with unique pathology (Cozzolino et al. 2008).

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 267

neurotoxic (Caughey and Lansbury 2003). The harmful nature of these oligomers compared to larger protein aggregates may be due to their lower stability, higher degree of solvent accessible surface area, and an increased tendency to form non-native associations with essential cellular components (Bucciantini et al. 2002; Knowles et al. 2007). For example, aggregates of many disease-associated proteins, including mutant SOD1, have been found to interact with the ubiquitin-proteasome system (Mouradian 2002; Sakamoto 2002; Urushitani et al. 2002; Valentine et al. 2005), folding chaperones (Bruening et al. 1999; Wyttenbach et al. 2000; Shinder et al. 2001; Okado-Matsumoto and Fridovich 2002), and the outer mitochondrial membrane (Vande Velde et al. 2008). These cellular components play central roles in regulating many critical cellular events ranging from cell division to apoptosis, and their impairment may represent common mechanisms by which aggregates of different proteins can cause cellular dysregulation and cell death (Hol and Scheper 2008; Gidalevitz et al. 2010). Many factors are involved in modulating protein aggregation, and

Globular protein folding begins on the ribosome, as newly synthesized, unstructured polypeptide chains start to make favourable intramolecular contacts (Dobson 2004). As it further folds into its mature, native state, a protein may populate multiple conformational or intermediate states, and undergo various co- and post-translational modifications (refer to Figure 1). The rate determining step of folding involves overcoming the major energetic barrier to folding by forming a transition state complex prior to attaining the native state. In more complex cases, protein folding can involve more than one energetic barrier (Dobson 2004). Other proteins are unable to adopt a stable, well folded structure and exist as an ensemble of fluctuating, poorly structured conformations (Uversky and Dunker 2010). Thermodynamic protein stability is defined as the difference in energy between the denatured, unfolded state and the native, folded state. If there is a large separation in energy between the unfolded and folded states, the protein has high global thermodynamic stability. Stability can also be assessed by the rate of native protein unfolding, which determines how long the polypeptide remains in the folded state. This is referred to as kinetic stability, defined as the difference in energy between the folded conformation and the transition state. The closer these species are in energy, the higher the rate of unfolding

In general, both thermodynamic and kinetic destabilization of proteins by chemical modifications or by mutations favours global protein unfolding and exposure of the hydrophobic groups that are normally buried in the protein core. This can promote the formation of non-native intermolecular contacts between proteins and the formation of aggregates. However, even subtle decreases in global protein stability are often accompanied by local destabilization and recent investigations have provided evidence for aggregate formation from native-like species (N\*, Figure 1) that have undergone much more restricted unfolding (Nelson and Eisenberg 2006; Chiti and Dobson 2009). Examples of aggregate formation from native-like states include various proteins associated with disease such as mutant lysozyme (Chiti and Dobson 2009), β2-microglobulin (Chiti and Dobson 2009), and SOD1 (Hwang et al. 2010). The propensity of a given globular protein to aggregate depends on how energetically feasible it is for the protein to access locally, partially or fully unfolded aggregation-prone state(s). Protein folding generally occurs in a

are surveyed in the following sections.

**1.2.1 Protein folding, stability and aggregation** 

and the lower the kinetic stability (Figure 1A).

Currently, there are two prevailing hypotheses for the toxic gain of SOD1 function that is observed in ALS: 1) new toxic enzymatic activity, and 2) protein misfolding resulting in formation of toxic aggregates (Valentine et al. 2005; Andersen 2006; Pasinelli and Brown 2006; Cozzolino et al. 2008; Turner and Talbot 2008). Since toxic enzymatic activity can damage the protein and cause aggregation, and conversely aggregation may result in toxic activity, these two hypotheses are not mutually exclusive. Theories involving gain of toxic activity involve altered metal binding by SOD1, resulting in the generation of reactive oxygen species, such as damaging hydroxyl and peroxynitrite radicals (Kurahashi et al. 2001; Alvarez et al. 2004). Alternatively, there is extensive evidence that ALS belongs to a growing group of protein misfolding diseases (Valentine et al. 2005; Chiti and Dobson 2006; Turner and Talbot 2008; Chiti and Dobson 2009; Deng et al. 2011). Protein inclusions, or aggregates, observed in the motor neurons and glial cells stain immunopositive for SOD1 in SOD1-linked fALS and some sALS patients (Kato et al. 2000; Liu et al. 2009; Forsberg et al. 2011) and are observed in mutant SOD1 animal models of ALS (Bruijn et al. 1998; Johnston et al. 2000). Thus, a major hypothesis in the field of ALS research is that SOD1 mutations decrease protein stability, alter protein folding and metal binding, and/or cause changes in other biophysical properties of the protein, resulting in an increased propensity of mutant SOD1 to form neurotoxic aggregates (Valentine et al. 2005).

Many reviews have summarized extensive investigations into the role of SOD1 in ALS, including *in vivo* mutant SOD1 models of ALS pathogenesis and their clinical implications (Bruijn et al. 2004; Boillee et al. 2006; Mitchell and Borasio 2007; Cozzolino et al. 2008), the numerous genetic elements and complex disease etiology associated with sALS and fALS (Boillee et al. 2006; Vucic and Kiernan 2009; Bastos et al. 2011), the various ALS rodent models used to study the underlying genetics and cause of motor neuron death in ALS (Van Den Bosch 2011), and the biophysical properties of mutant SOD1 in relation to possible disease mechanisms (Valentine et al. 2005). In this chapter we review recent research characterizing the stability, folding and misfolding, and the physical characteristics and mechanisms governing aggregation of mutant SOD1 *in vitro*. We describe in detail studies that reflect our own research and interests, but also include references to related work, to which we refer the interested reader. We will first review the general principles of protein stability and aggregation, which are pertinent to protein conformational diseases in general. Following this overview, we examine recent research that has characterized folding and aggregation of SOD1 and the relevance of this work to ALS.

### **1.2 Characteristics of protein aggregation**

Protein aggregation is a common phenomenon observed in both normal and abnormal physiological processes, and has been studied extensively for more than 30 years (Chiti and Dobson 2006). While protein association reactions are highly regulated and essential for cellular function, unregulated protein association causes a wide range of diseases, such as sickle-cell anaemia, serpinopathies, and, in particular, many neurodegenerative diseases including prion, Parkinson's, Alzheimer's, and Huntington's diseases (Chiti and Dobson 2006; Eisenberg et al. 2006; Chiti and Dobson 2009). These protein misfolding diseases are characterized by the formation of insoluble proteinacious deposits (aggregates) (Chiti and Dobson 2006), and the mechanisms and biological effects of aggregation in different diseases are an area of active research. In some cases, toxicity may be caused by large protein aggregates; however, smaller oligomeric protein species are generally considered more

Currently, there are two prevailing hypotheses for the toxic gain of SOD1 function that is observed in ALS: 1) new toxic enzymatic activity, and 2) protein misfolding resulting in formation of toxic aggregates (Valentine et al. 2005; Andersen 2006; Pasinelli and Brown 2006; Cozzolino et al. 2008; Turner and Talbot 2008). Since toxic enzymatic activity can damage the protein and cause aggregation, and conversely aggregation may result in toxic activity, these two hypotheses are not mutually exclusive. Theories involving gain of toxic activity involve altered metal binding by SOD1, resulting in the generation of reactive oxygen species, such as damaging hydroxyl and peroxynitrite radicals (Kurahashi et al. 2001; Alvarez et al. 2004). Alternatively, there is extensive evidence that ALS belongs to a growing group of protein misfolding diseases (Valentine et al. 2005; Chiti and Dobson 2006; Turner and Talbot 2008; Chiti and Dobson 2009; Deng et al. 2011). Protein inclusions, or aggregates, observed in the motor neurons and glial cells stain immunopositive for SOD1 in SOD1-linked fALS and some sALS patients (Kato et al. 2000; Liu et al. 2009; Forsberg et al. 2011) and are observed in mutant SOD1 animal models of ALS (Bruijn et al. 1998; Johnston et al. 2000). Thus, a major hypothesis in the field of ALS research is that SOD1 mutations decrease protein stability, alter protein folding and metal binding, and/or cause changes in other biophysical properties of the protein, resulting in an increased propensity of mutant

Many reviews have summarized extensive investigations into the role of SOD1 in ALS, including *in vivo* mutant SOD1 models of ALS pathogenesis and their clinical implications (Bruijn et al. 2004; Boillee et al. 2006; Mitchell and Borasio 2007; Cozzolino et al. 2008), the numerous genetic elements and complex disease etiology associated with sALS and fALS (Boillee et al. 2006; Vucic and Kiernan 2009; Bastos et al. 2011), the various ALS rodent models used to study the underlying genetics and cause of motor neuron death in ALS (Van Den Bosch 2011), and the biophysical properties of mutant SOD1 in relation to possible disease mechanisms (Valentine et al. 2005). In this chapter we review recent research characterizing the stability, folding and misfolding, and the physical characteristics and mechanisms governing aggregation of mutant SOD1 *in vitro*. We describe in detail studies that reflect our own research and interests, but also include references to related work, to which we refer the interested reader. We will first review the general principles of protein stability and aggregation, which are pertinent to protein conformational diseases in general. Following this overview, we examine recent research that has characterized folding and

Protein aggregation is a common phenomenon observed in both normal and abnormal physiological processes, and has been studied extensively for more than 30 years (Chiti and Dobson 2006). While protein association reactions are highly regulated and essential for cellular function, unregulated protein association causes a wide range of diseases, such as sickle-cell anaemia, serpinopathies, and, in particular, many neurodegenerative diseases including prion, Parkinson's, Alzheimer's, and Huntington's diseases (Chiti and Dobson 2006; Eisenberg et al. 2006; Chiti and Dobson 2009). These protein misfolding diseases are characterized by the formation of insoluble proteinacious deposits (aggregates) (Chiti and Dobson 2006), and the mechanisms and biological effects of aggregation in different diseases are an area of active research. In some cases, toxicity may be caused by large protein aggregates; however, smaller oligomeric protein species are generally considered more

SOD1 to form neurotoxic aggregates (Valentine et al. 2005).

aggregation of SOD1 and the relevance of this work to ALS.

**1.2 Characteristics of protein aggregation** 

neurotoxic (Caughey and Lansbury 2003). The harmful nature of these oligomers compared to larger protein aggregates may be due to their lower stability, higher degree of solvent accessible surface area, and an increased tendency to form non-native associations with essential cellular components (Bucciantini et al. 2002; Knowles et al. 2007). For example, aggregates of many disease-associated proteins, including mutant SOD1, have been found to interact with the ubiquitin-proteasome system (Mouradian 2002; Sakamoto 2002; Urushitani et al. 2002; Valentine et al. 2005), folding chaperones (Bruening et al. 1999; Wyttenbach et al. 2000; Shinder et al. 2001; Okado-Matsumoto and Fridovich 2002), and the outer mitochondrial membrane (Vande Velde et al. 2008). These cellular components play central roles in regulating many critical cellular events ranging from cell division to apoptosis, and their impairment may represent common mechanisms by which aggregates of different proteins can cause cellular dysregulation and cell death (Hol and Scheper 2008; Gidalevitz et al. 2010). Many factors are involved in modulating protein aggregation, and are surveyed in the following sections.

### **1.2.1 Protein folding, stability and aggregation**

Globular protein folding begins on the ribosome, as newly synthesized, unstructured polypeptide chains start to make favourable intramolecular contacts (Dobson 2004). As it further folds into its mature, native state, a protein may populate multiple conformational or intermediate states, and undergo various co- and post-translational modifications (refer to Figure 1). The rate determining step of folding involves overcoming the major energetic barrier to folding by forming a transition state complex prior to attaining the native state. In more complex cases, protein folding can involve more than one energetic barrier (Dobson 2004). Other proteins are unable to adopt a stable, well folded structure and exist as an ensemble of fluctuating, poorly structured conformations (Uversky and Dunker 2010). Thermodynamic protein stability is defined as the difference in energy between the denatured, unfolded state and the native, folded state. If there is a large separation in energy between the unfolded and folded states, the protein has high global thermodynamic stability. Stability can also be assessed by the rate of native protein unfolding, which determines how long the polypeptide remains in the folded state. This is referred to as kinetic stability, defined as the difference in energy between the folded conformation and the transition state. The closer these species are in energy, the higher the rate of unfolding and the lower the kinetic stability (Figure 1A).

In general, both thermodynamic and kinetic destabilization of proteins by chemical modifications or by mutations favours global protein unfolding and exposure of the hydrophobic groups that are normally buried in the protein core. This can promote the formation of non-native intermolecular contacts between proteins and the formation of aggregates. However, even subtle decreases in global protein stability are often accompanied by local destabilization and recent investigations have provided evidence for aggregate formation from native-like species (N\*, Figure 1) that have undergone much more restricted unfolding (Nelson and Eisenberg 2006; Chiti and Dobson 2009). Examples of aggregate formation from native-like states include various proteins associated with disease such as mutant lysozyme (Chiti and Dobson 2009), β2-microglobulin (Chiti and Dobson 2009), and SOD1 (Hwang et al. 2010). The propensity of a given globular protein to aggregate depends on how energetically feasible it is for the protein to access locally, partially or fully unfolded aggregation-prone state(s). Protein folding generally occurs in a

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 269

the aggregation propensity of a protein (Chiti et al. 2003). Several studies have suggested that SOD1 mutations promote exposure of hydrophobic regions that can promote aggregation (Tiwari et al. 2009; Munch and Bertolotti 2010). Taken together, these studies indicate that aggregation is at least partially controlled by the physicochemical properties of amino acid residues within a polypeptide sequence (Chiti et al. 2003; Tartaglia et al. 2008). In addition, solution conditions can modulate the stability, conformation, and the intermolecular interactions of a protein in solution, and can thereby influence the rate of protein aggregation and the type of aggregate structure formed (Chi et al. 2003; Mahler et al. 2009). Importantly, variations in solution conditions can cause the same protein to aggregate by fundamentally different mechanisms (Goers et al. 2002; Vetri and Militello 2005; Necula et al. 2007). Temperature, pH, macromolecular crowding, agitation, and ionic strength are all variables that can influence aggregation (Chi et al. 2003; Munishkina et al. 2004; Mahler et al. 2009; Sicorello et al. 2009). A number of studies have used different solution conditions (increased temperature, decreased pH, increased ionic strength, sonication or agitation) to promote the formation of well-structured, fibrillar amyloid aggregates (see 1.2.3) by various forms of SOD1 (Stathopulos et al. 2004; Chattopadhyay et al. 2008; Chattopadhyay and Valentine 2009; Oztug Durer et al. 2009). Other studies have demonstrated soluble oligomer and small aggregate formation by various forms of SOD1 in quiescent, physiologically relevant solution conditions (Vassall, 2011, Hwang, 2010, Banci, 2008). Thus, it is evident that multiple factors can greatly influence protein folding and aggregation and these factors must be considered when investigating the molecular mechanisms of protein aggregation.

Protein aggregation is a general term that describes a number of diverse processes that culminate in the formation of non-native, multimeric complexes of varied conformations. These aggregates can range from small, soluble oligomers, to larger amorphous structures, and insoluble, well-structured fibrils (Uversky and Dunker 2010). Amyloid is a common, well characterized, type of aggregate formed by proteins associated with many diseases, including the neurodegenerative prion, Parkinson's, Alzheimer's, and Huntington's diseases (Chiti and Dobson 2006; Chiti and Dobson 2009). Extensive studies of amyloid have resulted in significant advances in understanding the underlying molecular basis of protein aggregation (Sipe and Cohen 2000; Chiti and Dobson 2006; Eisenberg et al. 2006; Chiti and Dobson 2009). Classically defined amyloid is characterized by an unbranched, fibrillar aggregate morphology, which exhibits green-gold birefringence upon binding Congo red (Sipe and Cohen 2000), a dye used in disease diagnosis, and a cross-β x-ray diffraction pattern due to the presence of β-strands oriented perpendicular to the long axis of the fibre (Serpell 2000). These large aggregates can be extremely stable and unaffected by cellular clearance machinery (Dobson 1999; Knowles et al. 2007). There is extensive evidence that most and perhaps all proteins can form amyloid under suitable, typically destabilizing, conditions (Dobson 1999; Munishkina et al. 2004; Stathopulos et al. 2004). Amyloid formation can arise from association of unstructured, partially folded, or native-like species, and can be prevented by factors that favour native folding (Chiti and Dobson 2009). These include such factors as: interactions with molecular chaperones that can stabilize partially folded conformations and increase the folding rate; and post-translational modifications or ligand binding that can stabilize the native state and prevent unfolding (Dobson 2004; Chiti and Dobson 2009). Protein size is also a factor that modulates the propensity of a protein to

**1.2.3 Amyloid formation** 

cooperative fashion with minimal formation of partially folded species and this cooperativity generates a sufficiently large energy barrier between the unfolded and folded states, which decreases the likelihood of unfolding and aggregation (Dobson 1999; Dobson 2004; Tartaglia et al. 2008).

Fig. 1. Protein stability, misfolding and aggregation.

In panel A, the difference between thermodynamic and kinetic stability is shown. U, TS‡, and N refer to the unfolded, transition and native states of the protein, respectively. Refer to the main text for further explanation. In panel B, the effects of native state (N) destabilization by mutation on the population of locally unfolded, native-like (N\*), partially folded intermediate (I) and fully unfolded (U) states is shown. Aggregation may occur from N\*, I or U, and the morphology of the aggregates formed may depend on the conformation of the protein prior to aggregation. Mutations that destabilize N, decrease the energy difference between the N and the more unfolded states (N\*, I or U), and thereby promote aggregation. Note that destabilization of N, does not necessarily imply destabilization of I. Mutations that destabilize N, may stabilize or destabilize I, resulting in a large increase or decrease, respectively, in the population of I compared to levels observed in the native folding pathway. Panel B was adapted from (Chiti and Dobson 2009).

### **1.2.2 Factors that modulate aggregation of polypeptides**

In addition to protein stability and structure, many other factors, such as physicochemical properties of amino acids within a protein sequence and solution conditions, can affect protein aggregation. Hydrophobicity, β-sheet propensity, and charge of a polypeptide sequence have been shown to modulate the formation of amyloid aggregates (refer to section 1.2.3) by unfolded proteins (Chiti et al. 2003). Interestingly, these properties are also important for facilitating correct protein folding, suggesting that while similar forces contribute to both processes, different key residues are involved in forming the initial contacts that drive native protein folding and aggregation (Jahn and Radford 2008). In many cases, the overall aggregation propensity of a protein increases if the primary sequence contains short stretches of amino acids with properties that favour aggregation, for example, low net charge, extensive hydrophobicity, and/or a tendency to form a β-sheet over an αhelix (Tartaglia et al. 2008). Interestingly, many fALS-associated SOD1 mutations decrease the net charge of the protein, which may promote aggregation and explain why certain mutations give rise to a shorter disease duration (Sandelin et al. 2007; Shaw and Valentine 2007; Bystrom et al. 2010). Sequence hydrophobicity also plays a large role in modulating

cooperative fashion with minimal formation of partially folded species and this cooperativity generates a sufficiently large energy barrier between the unfolded and folded states, which decreases the likelihood of unfolding and aggregation (Dobson 1999; Dobson

In panel A, the difference between thermodynamic and kinetic stability is shown. U, TS‡, and N refer to the unfolded, transition and native states of the protein, respectively. Refer to

destabilization by mutation on the population of locally unfolded, native-like (N\*), partially folded intermediate (I) and fully unfolded (U) states is shown. Aggregation may occur from N\*, I or U, and the morphology of the aggregates formed may depend on the conformation of the protein prior to aggregation. Mutations that destabilize N, decrease the energy difference between the N and the more unfolded states (N\*, I or U), and thereby promote aggregation. Note that destabilization of N, does not necessarily imply destabilization of I. Mutations that destabilize N, may stabilize or destabilize I, resulting in a large increase or decrease, respectively, in the population of I compared to levels observed in the native

In addition to protein stability and structure, many other factors, such as physicochemical properties of amino acids within a protein sequence and solution conditions, can affect protein aggregation. Hydrophobicity, β-sheet propensity, and charge of a polypeptide sequence have been shown to modulate the formation of amyloid aggregates (refer to section 1.2.3) by unfolded proteins (Chiti et al. 2003). Interestingly, these properties are also important for facilitating correct protein folding, suggesting that while similar forces contribute to both processes, different key residues are involved in forming the initial contacts that drive native protein folding and aggregation (Jahn and Radford 2008). In many cases, the overall aggregation propensity of a protein increases if the primary sequence contains short stretches of amino acids with properties that favour aggregation, for example, low net charge, extensive hydrophobicity, and/or a tendency to form a β-sheet over an αhelix (Tartaglia et al. 2008). Interestingly, many fALS-associated SOD1 mutations decrease the net charge of the protein, which may promote aggregation and explain why certain mutations give rise to a shorter disease duration (Sandelin et al. 2007; Shaw and Valentine 2007; Bystrom et al. 2010). Sequence hydrophobicity also plays a large role in modulating

the main text for further explanation. In panel B, the effects of native state (N)

folding pathway. Panel B was adapted from (Chiti and Dobson 2009).

**1.2.2 Factors that modulate aggregation of polypeptides** 

2004; Tartaglia et al. 2008).

Fig. 1. Protein stability, misfolding and aggregation.

the aggregation propensity of a protein (Chiti et al. 2003). Several studies have suggested that SOD1 mutations promote exposure of hydrophobic regions that can promote aggregation (Tiwari et al. 2009; Munch and Bertolotti 2010). Taken together, these studies indicate that aggregation is at least partially controlled by the physicochemical properties of amino acid residues within a polypeptide sequence (Chiti et al. 2003; Tartaglia et al. 2008). In addition, solution conditions can modulate the stability, conformation, and the intermolecular interactions of a protein in solution, and can thereby influence the rate of protein aggregation and the type of aggregate structure formed (Chi et al. 2003; Mahler et al. 2009). Importantly, variations in solution conditions can cause the same protein to aggregate by fundamentally different mechanisms (Goers et al. 2002; Vetri and Militello 2005; Necula et al. 2007). Temperature, pH, macromolecular crowding, agitation, and ionic strength are all variables that can influence aggregation (Chi et al. 2003; Munishkina et al. 2004; Mahler et al. 2009; Sicorello et al. 2009). A number of studies have used different solution conditions (increased temperature, decreased pH, increased ionic strength, sonication or agitation) to promote the formation of well-structured, fibrillar amyloid aggregates (see 1.2.3) by various forms of SOD1 (Stathopulos et al. 2004; Chattopadhyay et al. 2008; Chattopadhyay and Valentine 2009; Oztug Durer et al. 2009). Other studies have demonstrated soluble oligomer and small aggregate formation by various forms of SOD1 in quiescent, physiologically relevant solution conditions (Vassall, 2011, Hwang, 2010, Banci, 2008). Thus, it is evident that multiple factors can greatly influence protein folding and aggregation and these factors must be considered when investigating the molecular mechanisms of protein aggregation.
