Do Proteins Always Benefit from a Stability Increase? Relevant and Residual Stabilisation in a Three-state Protein by Charge Optimisation

Structural Analysis of an Equilibrium Folding Intermediate in the Apoflavodoxin Native Ensemble by Small-Angle X-ray Scattering

“…One such mutant is E20K/E72K, which combines two previously reported mutations that stabilize the intermediate. 47 According to our analysis, the E20K/E72K intermediate is significantly stabilized compared to the unfolded state (transition temperatures of 40.6°C and 65.7°C), and its molar fraction is quite high (0.83 at 55.4°C; unpublished results). A second apoflavodoxin variant (F98N) bears a destabilizing mutation at the putative interface between the native-like region and the unfolded region of the wild-type thermal intermediate.…”

“…The thermal stability of the apo form has been described in detail elsewhere. 43,47 The occurrence of an equilibrium intermediate in thermal unfolding is evidenced by the noncoincidence of thermal unfolding curves monitored using various spectroscopic techniques. The tryptophan fluorescence emission, near-UV absorbance, far-UV circular dichroism (CD), and near-UV CD curves do not superimpose, but they can be simultaneously fitted to a three-state model (Fig.…”

“…27,43,47,58,59 From these, a qualitative picture of the thermal intermediate ensemble can be derived by interpreting that the environment of residues with small ϕ-values is structurally perturbed in the intermediate structure with respect to the native one. A higher-resolution structure of the thermal intermediate can be derived by incorporating these data into multiple restrained MD (rMD) simulations, a strategy that has been widely used to describe the transition states of folding.…”

Flavodoxins: sequence, folding, binding, function and beyond

“…The stability of apoflavodoxin has also been investigated by chemical denaturation, but in this case no common scheme has emerged: while for some apoflavodoxins an intermediate has been described [22], for others, a simple two-state mechanism can explain the chemical unfolding [20]. The apoflavodoxin from Anabaena has additionally been used as a model protein to investigate general stability principles (such as the contribution of cation/pi interactions [28], charge/charge interactions [29][30] and surface-exposed hydrogen bonds [31] to protein stability), to put forward new concepts such as those of the relevant and residual stabilities of proteins with equilibrium intermediates [32], and to obtain structural and thermodynamic information on molten globule intermediates [33]. The first three-dimensional structure of an apoflavodoxin (that from Anabaena) [34] revealed that the fold of the apoprotein was essentially identical to that of the functional holoflavodoxin complex [35], the only significant differences being traced to one of the FMN binding loops (57-63) that appears displaced so as to close the gap left by the absent cofactor (Fig.…”

The Flavodoxin from Helicobacter pylori: Structural Determinants of Thermostability and FMN Cofactor Binding

“…In fact, the native state is by large the most stabilized state upon FMN binding, which increases the energy gap with the first intermediate without hardly modifying the two subsequent transitions. The fact that FMN binding increases the relevant stability of the protein (N ↔ I 1 equilibrium) (44), while leaving the residual stability of the partly unfolded conformations (I 1 ↔ I 2 ↔ D) unchanged is consistent with I 1 displaying a structure similar to that characterized for the single thermal intermediate that appears in the thermal unfolding of the apoflavodoxin from Anabaena (23). That in H. pylori flavodoxin the binding of the cofactor does not lead to a highly cooperative two-state unfolding is simply due to the lower relevant stability and the higher residual one of the H. pylori apoprotein, compared to the Anabaena one, together with the lower affinity of FMN binding.…”