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The intermediate state detected at low denaturant concentration in the case of wtCLIC1 at pH 5.5 is more structured and stable than the molten-globule state detected in the unfolding transitions of CLIC1-M32A at pH 7.0. In terms of secondary structural content, wtCLIC1 at pH 5.5 in 3.8 M∑ urea shows a ~ 25 % decrease in its far-UV CD signal as compared to the spectrum of wtCLIC1 at pH 5.5 in the absence of denaturant (McIntyre, 2006). On the other hand, the far-UV-CD spectrum of CLIC1-M32A at pH 7.0 in 3.4 MΣ urea displays ~ 50 % loss in ellipticity relative to the spectrum of native CLIC1-M32A at pH 7.0 (Figure 41). Therefore, the molten- globule state of CLIC1-M32A has lost approximately double the helical content as compared to the molten-globule state of wtCLIC1. This is confirmed by pulse- labelling DXMS where the conversion from native to intermediate state involves ~ 31 and 41 % loss in structural content, i.e. helices/sheets/coils, in the case of wtCLIC1 and CLIC1-M32A, respectively. In addition, ANS binding studies show that the wild- type intermediate has fewer exposed surfaces than the CLIC1-M32A intermediate since its bound-ANS signal is approximately 2.5 times lower than in the case of the mutant. Furthermore, the λem max of ANS bound to wtCLIC1 is blue shifted compared

to the λem max of ANS bound to CLIC1-M32A (460 nm and 470 nm, respectively).

This means that the exposed surfaces are more hydrophobic in the wild-type protein

suggesting a more structured intermediate. In summary, the molten-globule state of wtCLIC1 in comparison to the CLIC1-M32A intermediate:

(1) is more stable since it exists at higher urea concentration.

(2) has preserved more of its secondary and tertiary structural content.

(3) has fewer exposed hydrophobic surfaces.

(4) is more structured.

Similarly to wtCLIC1, the fluorescence spectrum of the molten-globule state of CLIC1-M32A exhibits a blue shift in its λem max relative to the spectrum of native

CLIC1-M32A (McIntyre, 2006; section 3.7.4.1 and Figure 40). This suggests that Trp35 is more buried in the intermediate state than in the native state. In the case of wtCLIC1, the burial of tryptophan was attributed to the formation of an oligomeric structure (McIntyre, 2006). If this is true than pulse-labelling DXMS experiments, performed in this study, should show an increase in the number of residues protected from deuterium exchange as an oligomeric state forms at low denaturant concentrations. However, no such evidence could be found both for wtCLIC1 and CLIC1-M32A (Figure 46 B and C, Appendix Table H). Hence, the burial of Trp35 is attributed to local structural changes as the protein unfolds instead of global rearrangement and/or complex formation.

Which regions of wtCLIC1 and CLIC1-M32A unfold during the conversion from a native state to a molten-globule state (N → I)? This is a difficult question to answer since most of the probes used in this study provide global information and as such they do not present direct evidence on local structural changes. However, there are a number of clues to which structures participate in the N → I conversion. Continuous labelling DXMS indicates that the C-terminal domain of wtCLIC1 and CLIC1-M32A is more stable than the N-terminal domain (Nathaniel, 2006; Figure 31). Hence, the first structures to be perturbed during the N → I conversion will be the helices/sheets/coils in the N-domain. Far-UV-CD showed that 25 % and 50 % helical content is lost in the N → I conversion for wtCLIC1 and CLIC1-M32A, respectively (McIntyre, 2006; Figure 41). The N-terminal domain of CLIC1 contains 3 helices that make up approximately a quarter of the total helical content. Therefore, in the case of wtCLIC1 the N → I conversion possibly involves the re-structuring of the N-domain

helices. This is probably also true for CLIC1-M32A since continuous labelling DXMS showed that the Met32Ala mutation did not affect the N-domain of CLIC1 (Figure 32). If so, approximately another 25 % helical structures unfold upon N → I conversion of CLIC1-M32A. Continuous labelling DXMS shows that h8 – h9 region becomes more flexible as a result of the Met32Ala mutation (Figure 32). The destabilization of this region in the N state, due to removal of tertiary contacts, implies that h8, h9 and possibly h7 will be one of the first structures to be disturbed as the protein starts unfolding. These 3 helices form roughly 25 % of the total helical content. Therefore, it seems that in addition to three N-domain helices (h1, h2, h3) three C-domain helices (h7, h8, h9), in total half the helical content of CLIC1, unfold upon conversion of CLIC1-M32A from N → I. This is supported by the fact that the Cm value of the far-UV-CD unfolding curve coincides with the first phase of the

fluorescence unfolding curves of CLIC1-M32A (Figure 39). Hence, the equilibrium intermediate has approximately half of the secondary structural content of the native state. A third line of evidence is provided by pulse-labelling DXMS experiments where the number of incorporated deuteriums is directly related to the number of residues that become unstructured as the protein unfolds. The transition from N to I of wtCLIC1 results in approximately 30 % increase in unprotected residues (Table 4). On the other hand, upon conversion of CLIC1-M32A from N → I, 40 % of the amide hydrogens became unprotected (Table 4). The ~ 10 % difference in structures that unfold between the I state of wtCLIC1 and the I state of CLIC1-M32A can be accounted by destabilization of the region encompassing helices 7 – 9, which in total forms roughly 10 % of the total structural content of CLIC1. Hence, apart from the additional h7 – h9 region, the same structures unfold during the N → I conversion in wtCLIC1 and CLIC1-M32A. In must be noted that the above proposal is oversimplified in that it does not take into account any structures that may refold in the conversion and hence generate new helices and/or sheets. This is very possible especially in the case of the proposed TMD-s2 that has a high helical propensity which upon N-domain unfolding may convert to a helix (Nathaniel, 2006). However, as a rough guideline, this rearrangement model indicates that the Met32Ala mutation and acidic pH affect the conversion of CLIC1 from N to I in the same way. And so, the unfolding of the N-terminal domain of wtCLIC1 probably involves the uncoupling of the conserved inter-domain lock-and-key motif. In order to confirm this future work should make use of continuous- as well as pulse-labelling DXMS followed by

pepsin digestion to compare the native and intermediate states of wtCLIC1 and CLIC1-M32A. As performed in this study on native CLIC1-M32A and CLIC1-E81M (sections 2.2.12.1 and 3.6), pepsin digestion will provide more local information through the generation of peptide fragments. Hence, local structural changes will be detected during the conversion of CLIC1 from native to intermediate state.

According to data obtained from pulse-labelling DXMS, the I state is stabilized at low denaturant concentrations but present throughout the unfolding transition of wtCLIC1 at pH 5.5 and CLIC1-M32A at pH 7.0 (Figure 46). On the other hand, ANS binding studies indicate that no binding of the hydrophobic dye to these proteins occurs in the pre- and post-unfolding transition (McIntyre, 2006; Figure 42). A possible explanation for this discrepancy may be that the difference in protein concentration used in the two techniques is approximately 50 fold and so the I state is probably undetected at low and high denaturant concentrations by ANS fluorescence due to the low protein concentration used. If this is the case the detection of the I state in the absence of denaturant indicates that the drop in pH from 7.0 to 5.5 and the Met32Ala substitution have lowered the energy barrier between the N and I conformations so that the I state becomes more populated and hence detected via pulse-labelling DXMS. The decrease in energy gap between N and I is due to destabilization of the N conformation and/or stabilization of the I state. As the conditions shift toward the I conformation (i.e. mild denaturing conditions, drop in pH, reducing environment, increase in temperature) it becomes progressively more populated until a point where it is the predominant state [i.e native wtCLIC1 at pH 5.5/37 °C has exposed hydrophobic surfaces and looser packing with ~ 16 % less helical content than native wtCLIC1 at pH 7.0/20 °C (McIntyre, 2006)]. Some mutants such a CLIC1-M32A mimic the effects of the environment by destabilizing the N state and/or stabilizing the I conformation. In fact the replacement of Lys37, which is part of the h1 N-capping motif and forms contacts between h1 and h3 through a salt-bridge with Glu85, with threonine resulted in I being the pre-dominant state (McIntyre, 2006). This is an indication that interactions such as the inter-domain lock-and-key and the h1 N- capping motifs form part of the mechanism responsible for the transition of CLIC1 from a soluble state to a membrane-competent state.