N-acetyl amino acid amide solubility in aqueous 1,6-hexanediol solutions: Insights into the protein droplet deformation mechanism.

Proteinaceous liquid droplets, generated by liquid-liquid phase separation, function as membraneless compartments that are essential for diverse biological functions. Studies addressing droplet generation have used 1,6-hexanediol (1,6-HD) as a droplet-discerning agent owing to its capacity to induce droplet deformation. Despite the empirical utility of 1,6-HD, the mechanism underlying 1,6-HD-induced droplet deformation remains unknown. In this study, the solubilities of N-acetyl amino acid amides, which correspond to proteinogenic amino acid residues, were examined in the presence of 1,6-HD at 25 °C. Other solvents included ethanol, 1-propanol, and amides. Remarkably, 1,6-HD effectively solubilized hydrophobic species (particularly aromatic species) and exhibited reduced efficacy in solubilizing hydrophilic species and peptide bond moieties. These solubilizing effects are reflected in changes in protein solubility and structure. Specifically, 1,6-HD primarily targets the hydrophobic regions of a protein, increasing protein solubility without causing substantial structural changes. This solubilization mechanism is essential for elucidating the role of 1,6-HD as a droplet-discerning agent and recognizing its potential limitations.

Presence of ß-Turn Structure in Recombinant Spider Silk Dissolved in Formic Acid Revealed with NMR.

Spider dragline silk is a biopolymer with excellent mechanical properties. The development of recombinant spider silk protein (RSP)-based materials with these properties is desirable. Formic acid (FA) is a spinning solvent for regenerated Bombyx mori silk fiber with excellent mechanical properties. To use FA as a spinning solvent for RSP with the sequence of major ampullate spider silk protein from Araneus diadematus, we determined the conformation of RSP in FA using solution NMR to determine the role of FA as a spinning solvent. We assigned 1H, 13C, and 15N chemical shifts to 32-residue repetitive sequences, including polyAla and Gly-rich regions of RSP. Chemical shift evaluation revealed that RSP is in mainly random coil conformation with partially type II ß-turn structure in the Gly-Pro-Gly-X motifs of the Gly-rich region in FA, which was confirmed by the 15N NOE data. In addition, formylation at the Ser OH groups occurred in FA. Furthermore, we evaluated the conformation of the as-cast film of RSP dissolved in FA using solid-state NMR and found that ß-sheet structure was predominantly formed.

The solubility of N-acetyl amino acid amides in organic acid and alcohol solutions: Mechanistic insight into structural protein solubilization.

Structural proteins such as spider silk and silkworm silk are generally poorly soluble in aqueous and organic solutions, making them difficult to manipulate in manufacturing processes. Although some organic acids and alcohols, such as formic acid and hexafluoroisopropanol (HFIP), effectively solubilize poorly soluble proteins, little is known about their protein solubilization mechanism. In this study, the solubility of N-acetyl amino acid amide compounds in organic solvents-formic acid, acetic acid, HFIP and isopropanol-was measured to clarify the protein solubilization mechanism at the amino acid residue level. On the basis of thermodynamic analyses of the solubility in terms of the transfer free energy (from water to organic solvents), every organic solvent was found to be effective in thermodynamically stabilizing hydrophobic amino acid side chains in the liquid phase. Formic acid and HFIP were comparably effective in the stabilization of the polypeptide backbone, whereas acetic acid and isopropanol were ineffective. Therefore, the significant solubilizing effect of formic acid and HFIP on the structural proteins was attributed to their favorable interactions with hydrophobic amino acid side chains and with the polypeptide backbone of the proteins. The present findings are useful for the optimization of protein manipulation and amino acid sequence design.

Role of the membrane potential in mitochondrial protein unfolding and import.

Newly synthesized mitochondrial precursor proteins have to become unfolded to cross the mitochondrial membranes. This unfolding is achieved primarily by mitochondrial Hsp70 (mtHsp70) for presequence-containing precursor proteins. However, the membrane potential across the inner membrane (ΔΨ) could also contribute to unfolding of short-presequence containing mitochondrial precursor proteins. Here we investigated the role of ΔΨ in mitochondrial protein unfolding and import. We found that the effects of mutations in the presequence on import rates are correlated well with the hydrophobicity or ability to interact with import motor components including mtHsp70, but not with ΔΨ (negative inside). A spontaneously unfolded precursor protein with a short presequence is therefore trapped by motor components including mtHsp70, but not ΔΨ, which could cause global unfolding of the precursor protein. Instead, ΔΨ may contribute the precursor unfolding by holding the presequence at the inner membrane for trapping of the unfolded species by the import motor system.

The Cholesterol-dependent Cytolysin Membrane-binding Interface Discriminates Lipid Environments of Cholesterol to Support ß-Barrel Pore Insertion.

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of ß-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the ß-barrel pore.

Disulfide-bond scanning reveals assembly state and ß-strand tilt angle of the PFO ß-barrel.

Perfringolysin O (PFO), a bacterial cholesterol-dependent cytolysin, binds a mammalian cell membrane, oligomerizes into a circular prepore complex (PPC) and forms a 250-Å transmembrane ß-barrel pore in the cell membrane. Each PFO monomer has two sets of three short α-helices that unfold and ultimately refold into two transmembrane ß-hairpin (TMH) components of the membrane-embedded ß-barrel. Interstrand disulfide-bond scanning revealed that ß-strands in a fully assembled PFO ß-barrel were strictly aligned and tilted at 20° to the membrane perpendicular. In contrast, in a low temperature-trapped PPC intermediate, the TMHs were unfolded and had sufficient freedom of motion to interact transiently with each other, yet the TMHs were not aligned or stably hydrogen bonded. The PFO PPC-to-pore transition therefore converts TMHs in a dynamic folding intermediate far above the membrane into TMHs that are hydrogen bonded to those of adjacent subunits in the bilayer-embedded ß-barrel.