Probing the Conformation-Dependent Preferential Binding of Ethanol to Cationic Glycylalanylglycine in Water/Ethanol by Vibrational and NMR Spectroscopy

Abstract

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.

Glycine in Water Favors the Polyproline II State

“…The environment of the amide group can be experimentally probed by chemical shift measurements. Previously reported values for GAG [72,73] (Table S8) are consistent with an increased hydration of the amide group of residue 2 relative to residue 3 (Table S6, columns 3 and 4) as increased hydration produces upfield shift of the chemical shift [74]. The carbonyl oxygen of residue 1 forms significantly fewer HBs with water than carbonyl oxygens of residues 2 and 3 ( Table S7).…”

“…Although there is no experimental data on conformational ensemble of central glycine in GGG, Eker et al reported that, while alanine-based tripeptides favor pPII in water, their pPII content is significantly reduced in DMSO [75]. Consistent with the key role of water in pPII stabilization, the addition of ethanol to water was also shown to disfavor the pPII mesostate of guest alanine in cationic GAG [72,76].…”

The Role of Backbone Hydration of Poly(N-isopropyl acrylamide) Across the Volume Phase Transition Compared to its Monomer

“…NMR experiments probing chemical shifts of amide protons are indicators of the groups involvement with water and CO groups 63 , 69 , 70 . While beyond the scope of the present study, Hetero-Nuclear-Correlation-Spectroscopy (HSQC) experiments 71 probing the temperature dependence of J coupling on isotopically labeled polymers could provide additional information on the involvement of amide protons 72 , 73 in hydrogen bonding.…”

The impact of thermal history on the structure of glycylalanylglycine ethanol/water gels

“…However, several peptides without aromaticity have been shown to readily self‐assemble into fibril networks. For example, glycine‐alanine‐glycine (GAG) and glycine‐histidine‐glycine (GHG) self‐assemble into very long microfibrils under the right conditions 19–26 . More specifically, GAG forms fibrils in the presence of a critical fraction of ethanol that depends on peptide concentration 26 .…”

“…Recent experimental studies suggest the hypothesis that GAG's self‐assembly is initiated by the accumulation of GAG at the ethanol/water interface, with the protonated C‐terminal immersed in ethanol and the N‐terminal exposed to water 19 . The amide I′ region of the GAG fibrils exhibits a doublet with rather sharp bands at 1644 and 1670 cm −1 , which means that GAG does not self‐assemble into typical β‐sheet tapes or ribbons 19 . Note that β‐sheets are generally considered the fundamental building block of amyloid‐like fibrils 27,28 .…”