VERIFICACIÓN DE IDEA A DEFENDER

The folding pathway of BPTI, in terms of the combinations of disulfide bonds formed during folding, has been studied extensively. Early studies of folding intermediates were performed by Thomas Creighton in the 1970s (Creighton 1975), around the same time as Anfinsen and colleagues were developing some of the fundamental principles governing polypeptide self-assembly and protein folding (Anfinsen 1973; Anfinsen and Scheraga 1975). Meanwhile, a high resolution crystal structure of BPTI was revealed (Deisenhofer and Steigemann 1975). Different in vitro conditions effecting BPTI folding and unfolding were explored, including the use of denaturing agents (Creighton 1977; Creighton 1978; Creighton 1979; Creighton 1980) and the role that PDI may have in its folding was first explored (Creighton, Hillson et al. 1980). Throughout the 1980s, various biophysical techniques were used to characterise native BPTI and various conformations thought to be key to its folding pathway (Kosen, Creighton et al. 1980; Kosen, Creighton et al. 1981), showing that disulfide bonds can be used as effective probes for studying protein folding (Creighton 1986). This led to exploration of the folding pathway of other small proteins (Kaderbhai and Austen 1985; Pace and Creighton 1986). With well characterised intermediates now in place, attention turned to the kinetics of BPTI folding, in an attempt to establish the rate at which intermediates were formed (Goldenberg 1988). Other, less populated intermediate species of BPTI folding were also being investigated (Vanmierlo, Darby et al. 1991a). By this stage, a folding pathway for BPTI had been well established, with relative rates for each step in the process being estimated, Figure 1.12. It indicated that the optimum folding pathway necessitated the creation of non-native disulfide bonds and that isomerisation of these species was rate-limiting to establish the native structure.

Figure 1.12: Model of the productive BPTI folding pathway, showing major disulfide intermediates in their approximate conformations (Darby, Morin et al. 1995). The non- productive quasi-native species (5-55, 14-38) has been omitted. The relative rate of each step is indicated by the thickness of the appropriate arrowhead; the wider the arrowhead, the greater the rate in that direction.

Meanwhile another group was independently studying intermediates of BPTI folding (Oas and Kim 1988). Controversially, they proposed an alternative folding pathway model, suggesting that only native disulfide bonds were predominant (Weissman and Kim 1991), Figure 1.13.

The discrepancies between the two models arise due to the manner in which kinetic folding intermediates are trapped by quenching. Whilst Creighton and colleagues used chemical quenchers, which may be inaccessible to buried thiols, Weissman and colleagues used an acid quenching technique. The controversy over the predominance of native disulfide species came to a head

formation of non-native disulfides was never under dispute; rather, it was a debate about the dominant or transient nature of the non-native disulfide bonds in the BPTI folding pathway.

Figure 1.13: Alternative BPTI folding pathway model, emphasising the predominance of native disulfide bonds (Weissman and Kim 1991). The relative conversion rates between disulfide species are labelled. R is fully reduced BPTI; N is natively folded; N* is a kinetically trapped intermediate. Less populated species, including non-native intermediates, are not shown but are still required in transitions to the (30-51, 5-55) species.

Meanwhile, further characterisation of the conformational properties of intermediates involved the use of both one and two dimensional 1H NMR (Vanmierlo, Darby et al. 1991a; Vanmierlo, Darby et al. 1991b; Darby, Vanmierlo et al. 1992; Staley and Kim 1992; Vanmierlo, Darby et al. 1992) and an X-ray crystal structure (Eigenbrot, Randal et al. 1992). These revealed that many intermediates have a native-like structure, which perhaps contributes to the difficulty of sequential native disulfide formation, due to steric hindrance of buried cysteines (Goldenberg 1992). The effects of other ER resident proteins on the folding of BPTI was investigated (Zapun, Creighton et al. 1992; Creighton, Bagley et al. 1993), showing the influence that PDI may have on the rate limiting steps of the process (see below). Further NMR studies focused on the influence of side chains on both the folding and structure of BPTI, revealing the importance of some aromatic rings (Kemmink and Creighton 1993; Kemmink,

Vanmierlo et al. 1993; Mendoza, Jarstfer et al. 1994; Yu, Weissman et al. 1995; Zhang and Goldenberg 1997).

Evidence of (30-51) as the most stable first disulfide bond in BPTI folding was revealed (Darby and Creighton 1993). The structure and dynamics of this critical one disulfide intermediate was further refined with the use of 1H-15N HSQC NMR of isotopically labelled intermediates (Vanmierlo, Darby et al. 1993). This, together with an independent NMR study of the same intermediate (Staley and Kim 1994), showed clear evidence that, despite only one disulfide being present, a large proportion of the protein showed native-like structure, with the first 14 N- terminal residues remaining unfolded. However, structure was only observed at low temperatures. Likewise, very similar structures were identified for both the (30-51, 5-14) and (30-51, 5-38) non-native intermediates using 1H NMR (Vanmierlo, Kemmink et al. 1994). Interestingly, NMR of fully reduced and unfolded BPTI suggested that it retained some structure (Pan, Barbar et al. 1995). Structural studies of BPTI mutants using 13C NMR were also performed (Hansen, Lauritzen et al. 1995).

Reviews comparing the folding pathways of several different proteins reveal common features in the formation of native disulfide bonds (Creighton, Zapun et al. 1995; Creighton 1995a; Creighton 1995b). A more recent comparison is available that relates these folding pathways to current understanding of the in vivo apparatus of prokaryotic and eukaryotic thiol-disulfide exchange (Mamathambika and Bardwell 2008).