The term denaturation denotes the loss of native tertiary and/ or secondary

structure without change in molecular weight (Tanford, 1997). The unfolding process
at neutral pH appears to involve multiple sequential steps and associated nonnative intermediates (De la Fuente, Singh, & Hemar, 2002; Edwards &Jameson,
2014; de Wit, 2009). Heating drives the monomer-dimer equilibrium towards the
monomeric state (N), and subsequently a reversible conformational rearrangement
of monomers (the Tanford transition) exposes the free thiol Cys121 and a
hydrophobic patch, a state denoted R. A second change in monomer conformation
occurs next, this time irreversible (the S state), followed by aggregation via
disulphide bonding and hydrophobic interactions. This reaction scheme can be
summarised:
N2NRSaggregates
The demarcation of native from non-native species is complex, and different
instrumental techniques draw this line in different places (Loveday, 2016).