Crowding Alone Cannot Account for Cosolute Effect on Amyloid Aggregation

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed “macromolecular crowding”. Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD), we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol) increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation

Linear rate-equilibrium relations arising from ion channel-bilayer energetic coupling

Linear rate-equilibrium (RE) relations, also known as linear free energy relations, are widely observed in chemical reactions, including protein folding, enzymatic catalysis, and channel gating. Despite the widespread occurrence of linear RE relations, the principles underlying the linear relation between changes in activation and equilibrium energy in macromolecular reactions remain enigmatic. When examining amphiphile regulation of gramicidin channel gating in lipid bilayers, we noted that the gating process could be described by a linear RE relation with a simple geometric interpretation. This description is possible because the gating process provides a well-understood reaction, in which structural changes in a bilayer-embedded model protein can be studied at the single-molecule level. It is thus possible to obtain quantitative information about the energetics of the reaction transition state and its position on a spatial coordinate. It turns out that the linear RE relation for the gramicidin monomer-dimer reaction can be understood, and the quantitative relation between changes in activation energy and equilibrium energy can be interpreted, by considering the effects of amphiphiles on the changes in bilayer elastic energy associated with channel gating. We are not aware that a similar simple mechanistic explanation of a linear RE relation has been provided for a chemical reaction in a macromolecule. RE relations generally should be useful for examining how amphiphile-induced changes in bilayer properties modulate membrane protein folding and function, and for distinguishing between direct (e.g., due to binding) and indirect (bilayer-mediated) effects

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

Micronutrients such as siderophore-bound iron and vitamin B12 cross the outer membrane of Gram-negative bacteria through a group of 22-stranded β-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the β-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellular-facing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs