The conformational propensity of amino acid residues is determined by an intricate balance of peptide-solvent and solvent-solvent interactions. To explore how the systematic replacement of water by a cosolvent affects the solvation of both the amino acid backbone and side chains, we performed a combined vibrational spectroscopy and NMR study of cationic glycylalanylglycine (GAG) in different ethanol/water mixtures of between 0 and 42 mol percent ethanol. Classical model peptide N'-methylacetamide was used as a reference system to probe solvent-induced spectroscopic changes. The alanine residue of GAG in water is known to exhibit a very high propensity for polyproline II (pPII). Adding up to 30 mol % ethanol at room temperature leads only to minor changes in the Ramachandran distribution of alanine, which mostly changes within the individual conformational subspaces. A further increase in the ethanol fractions leads to a destabilization of pPII and a stabilization of β-strand conformations. At higher temperatures, different degrees of enthalpy-entropy compensations lead to a much stronger influence of ethanol on the peptide's conformational distribution. Ethanol-induced changes in chemical shifts and amide I wavenumbers strongly suggest that ethanol replaces water preferentially in the solvation shell of the polar C-terminal peptide group and of the alanine side chain, whereas the N-terminal group remains mostly hydrated. Furthermore, we found that ethanol interacts more strongly with the peptide if the latter adopts β-strand conformations. This leads to an unusual positive temperature coefficient for the chemical shift of the C-terminal amide proton. Our data suggests a picture in which GAG eventually accumulates at water-ethanol interfaces if the ethanol fractions exceed 0.3, which explains why the further addition of ethanol eventually causes self-aggregation and the subsequent formation of a hydrogel.
system (T3SS). P. aeruginosa's most cytotoxic T3SS effector, ExoU, is a potent patatin-like phospholipase with a catalytic serine-aspartate dyad that destroys host cell membranes once activated by non-covalent interaction with ubiquitin. Two crystal structures of ExoU in complex with its chaperone SpcU have been reported, however the activated conformational state of ExoU in the presence of ubiquitin remains unknown. Consequently, ExoU's mechanism of action remains poorly understood. This study focuses on elucidating the conformational dynamics, membrane interaction, and structure of sites near the catalytic serine upon interaction with a ubiquitin cofactor and membrane substrate. Site-directed spin labeling (SDSL) in conjunction with electron paramagnetic (EPR) spectroscopy was used to examine the motional dynamics of sites near the catalytic serine upon interaction with diubiquitin (diUb) and membranes. Membrane penetration of the same sites upon interaction with diUb and membranes was investigated using power saturation EPR spectroscopy in the presence of various paramagnetic relaxation agents. Changes in spin label motion and membrane penetration were observed for sites near the catalytic serine in the presence of diUb and membranes combined, but not in the presence of either diUb or membranes alone, suggesting that a synergistic interaction with both ubiquitin and membranes is necessary to form an active catalytic site. Supported by NIH grant GM114234.
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