This fast band reflects within-membrane, conformationrmation BAK, and is lost in the presence of BID BH3 peptide, consistent with unfolding and activation at the level of a monomeric species.
Supercharging reagents were mixed into solutions containing folded proteins during ESI using theta-glass emitters which resulted in higher charging and loss of heme for myoglobin, consistent with unfolding of the proteins in the ESI droplets induced by the supercharging reagents.
This suggests that the helix αB of bound pKID is more native-like than the helix αA upon KIX-binding and consistent with the unfolding kinetics and landscape analysis.
Superficially, these changes appear to be consistent with the unfolding of i-motif DNA.
After heating with 6 M guanidine hydrochloride, both unlabelled MsbA and MsbA MIANS displayed a substantial red shift in their emission maxima to 335 and 337 nm respectively, consistent with partial unfolding of the protein.
Moreover, CD spectra of isolated SAS-5 N-terminal (residues 2 122) or C-terminal (269 404) fragments showed no persistent secondary structure or cooperative thermal unfolding, consistent with similar previous analysis of the SAS-5 N-terminus (Shimanovskaya et al., 2013).
Inspection of the time series of the N-C distance at 10 pN (Fig. 3 C, left) and 20 pN (Fig. 3 C, right), however, reveal that the SB model is not consistent with the unfolding mechanism of the coarse-grain model.
GFP is known to show non-equilibrium behavior in denaturant-induced unfolding [27] (consistent with the unfolding transitions shifting towards lower Gdn-HCl concentrations at increased incubation time (cf. Fig. S6B and C)), so true free energies of unfolding cannot be deduced from unfolding transitions alone.
These results are consistent with the entire unfolding leg of the G93A chevron reflecting the dimer dissociation reaction, similar to the other variants and WT at high urea concentrations.
This motion mode is consistent with a local unfolding induced by the perturbation of the protein's energy landscape by the oxidative modification and with the observed variations in the secondary structure content.
This is consistent with the simultaneous unfolding of domains both at high force and in sharp contrast with experiments in the absence of Ibalizumab, in which we observed one step for each CD4 domain (Supporting Information, Figure 7).
