NMR investigation of ion-pair modulation of protein structure and dynamics and its relation to protein misfolding diseases

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NMR investigation of ion-pair modulation of protein structure and dynamics and their relation to protein stability

Publication . Silva, Micael Simões; Cabrita, Eurico; Figueiredo, Ângelo

Salt ions differ in their ability to salt out or salt in proteins from aqueous solutions. Their effects on protein stability are known to be connected to ion hydration, ion pairing and ion-specific interactions with the protein. In general, cations follow the Hofmeister series for protein stabilisation behaviour, while for anions this is only true for proteins where the backbone effect is stronger than that of the positively charged side chains. Since at low concentrations electrostatic effects are expected to be the most significant, ion-specific effects become dominant at high salt concentrations. However, the molecular details of how ions interact with proteins have not yet been fully understood. In this thesis, using different nuclear magnetic resonance (NMR) methodologies and ionic liquids (ILs) as models for the investigation of salt and/or ion-pair effects on protein stability, I prove that molecular mechanisms which result in protein stabilisation or destabilisation are opposed (in enthalpic and entropic terms). These mechanisms depend not only on physical and chemical nature of ions but also on the protein properties. The variety of combinations to form ILs allowed the investigation of the effects of choline glutamate ([Ch][Glu], stabiliser) or 1-butyl-3-methylimidazolium dicyanamide ([Bmim][dca], denaturant) on two model proteins with distinct stability and structural properties: GB1 (DGF→U ~ 7 kcal/mol) and drkN SH3 (DGF→U ~ 0 kcal/mol). The possibility of ion-specific interactions with the protein was studied and the changes of protein structure, diffusion, dynamics, and solvent exchange in the presence of IL were characterized. The data gathered for GB1 and drkN SH3 revealed that [Ch][Glu] at high concentrations (> 1 M) stabilises proteins, not only via electrostatic contacts, but also through a preferential accumulation mechanism at the protein surface, in agreement with an entropicdriven mechanism due to excluded-volume effects. At low IL concentrations, a preferential hydration of the protein is not entirely excluded, which could lead to an initial protein destabilisation. On the other hand, [Bmim][dca] denatures proteins by preferential and direct but unfavourable hydrophobic interactions. These interactions occur not only with the core of the folded state, but also with the unfolded state (slowing the folding process). This was revealed by structural and thermodynamic studies with drkN SH3 where it was possible to directly evaluate the perturbations on the unfolded state due to the slow exchange between the two states. This interaction leads to a stabilisation of a residual helical structure in the unfolded ensemble, which contradicts the random coil-like structure typically found in the presence of denaturing agents. The data gathered provided a thorough understanding of ILprotein interactions as well as the mechanism by which they can affect protein’s equilibrium thermodynamics and kinetics, illustrating the importance of the unfolded state and a possible impact in the rational design of solvents in biotechnological processes, for example, increasing not only the catalytic activity but also the enzyme thermostability in such media.

2021-12-10Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

Número da atribuição

PD/BD/128202/2016