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Aspartic acid side chain effect - experimental and theoretical insight

Introduction In the CID mass spectra of peptides containing Asp, Glu and oxidized Cys selective cleavage of the amide bond C-terminal is dominant (the “ aspartic acid effect” ). Despite several proposed mechanisms a detailed description of the reaction pathway is still missing. Recently it was demonstrated that gas-phase H/D exchange can be used to investigate the aspartic side chain effect. Such use of H/D exchange and interest to understand aspartic side chain effect motivates this study Methods The H/D exchanges in Asp and Glu were performed within a 3 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (Extrel FTMS 2001, Madison, USA) with D2O (99.8%, Aldrich, Milwaukee, USA) as deuteration reagent. Calculations for various structures were performed using B3LYP functional with 6− 31G(d) basis set and the G3(MP2)//B3LYP computational protocol. Heptapeptide backbone (six glycines and aspartic acid) were treated with two layer (QM:QM) ONIOM treatment. In the combined QM/QM treatment, the -CH2CO-Asp-NHCH2- part was described at the B3LYP/6-31G(d) level of theory while the rest of the peptide was described at the HF/3-21G level. Preliminary data H/D exchange site specific treatment and some additional theoretical calculations confirmed the ability of a side chain carboxylic group to initiate proton transfer along with a new bond formation either to the next or the same of its oxygens i.e. the possibility of thus initiating peptide bond selective cleavage (“ aspartic acid effect” ). B3LYP/6-31G(d) and G3(MP2)//B3LYP potential energy profiles of the “ aspartic acid effect” on a model system CH3CO-Asp-NHCH3 suggest a concerted one step mechanism of proton transfer from the side chain carboxylic group to the backbone amide group accompanied by bond formation (i.e. between deprotonated carboxylic group and C atom of the backbone carbonyl) and peptide bond cleavage as favorable. Combined QM/QM treatment of the “ aspartic acid effect” in α -helix and β -sheet heptapetides confirms such model system predictions.

aspartic acid effect; H/D exchange; FT ICR MS; DFT

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