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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Impact of Ionic Liquids on the Conformation of PeptidesStudied by HR-MAS NMR SpectroscopyAnnekathrin Richardt, Carmen Mrestani-Klaus, and Frank BordusaInstitute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg,Halle, 06120, GermanyIntroductionIonic liquids (ILs) have attracted a rapid use as popular solvents in chemistry andbiocatalysis as well. They exhibit melting points lower than 100°C, low vapor pressure andare mostly more viscous than classic solvents. In our research, we used ILs as reactionmedium additives in the protease-mediated synthesis of proteins [1]. In the attempts toexplain the altered enzyme activities on the basis of a hypothetical effect of ionic liquids onthe spatial structure of the biocatalyst or peptide and protein targets, the impact of ILs onthe cis/trans ratio of Xaa-Pro peptide bonds was studied by solvent jump experiments [2].Consequently, direct interactions between dissolved peptides and ILs have to beinvestigated in order to understand how ionic liquids influence the structure of peptides.For this purpose, conventional high-resolution 1 H NMR measurements of IL/peptidesystems were initially performed. However, these experiments did not lead to useful resultsdue to extremely low peptide signal resolution and line-broadening effects caused by thehigh IL viscosity. Therefore, HR-MAS (high-resolution magic angle spinning) NMRspectroscopy has been successfully applied here as efficient, powerful and IL-compatibleanalytical method to facilitate structural analysis of IL/peptide systems on a molecularlevel.Results and DiscussionThe present study was focused upon investigations of peptide/IL systems using the modeltetrapeptides Ala-Xaa-Pro-Phe (Xaa = Ala, Glu, Gly, Lys, Phe) that exhibit high water aswell as IL solubilities. As ionic liquids, 1-ethyl-3-methylimidazolium diethylphosphate[EMIM][Et 2 PO 4 ], 1,3-dimethylimidazolium dimethylphosphate [MMIM][Me 2 PO 4 ] and1-ethyl-3-methylimidazolium trifluoroacetate [EMIM][CF 3 CO 2 ] were chosen (Figure 1).In general, imidazolium-type ILs arefrequently used, most investigated,commercially available and well watermiscible. They represent ideal model systemsfor structural investigations using NMRspectroscopy since imidazolium ring protonsare highly sensitive to the chemicalenvironment.In order to get evidence of directinteractions between the ILs and peptides aswell as of IL-induced conformational changesof the dissolved peptides, we utilized protonchemical shift difference values (Δδ).Accordingly, high-resolution NMR spectra ofthe peptides and of the ILs were recordedseparately in aqueous solution as a point ofreference. Afterwards, NMR spectra ofdifferent IL/peptide mixtures were acquired.Complete resonance assignment of both theFig. 1. Structures of the ionic liquids:a)[MMIM][Me 2 PO 4 ] b)[EMIM][Et 2 PO 4 ]c)[EMIM][CF 3 CO 2 ].ILs and the peptides as well as of theIL/peptide systems was done by applyingconventional two-dimensional NMRtechniques (Figure 2a). The proton chemicalshifts were extracted from the spectra as basisfor the determination of chemical shift differences between the reference systems inaqueous solution and the IL/peptide systems. cis/trans peptide isomer ratios weredetermined via integration of well-resolved signals in the 1 H NMR spectra.16