Real-Time 1H NMR reveals position and sequence dependences of amino acid isomerization in amyloid beta fragments in situ

Abstract

Amino acid isomers and their biologically relevant functions have been found in a variety of peptides and proteins in a living system. It is challenging, however, to specify how such isomerization proceeds at each amino acid independently in situ. In this work, the amino acid isomerization was quantified simultaneously at multiple residues in real time, by using solution-state 1H NMR in situ. The ability of NMR to distinguish isomerization product and reactant was demonstrated at different positions of aspartyl (Asp) residues in amyloid beta (Aβ) 1–40 fragments, as well as the glutamyl (Glu) residue. This enabled us to compare the isomerization of Asp1, Asp7, and Asp23, and Glu3 systematically in various Aβ fragments, in terms of the kinetics of isomerization in real time. It was found that Asp1 and Asp7 were prone to convert to iso-aspartyl (isoAsp) form, although Asp23 and Glu3 showed limited reactivity. The analysis demonstrated the role of histidine (His) in regulating Asp isomerization; His6 and His13/14 showed an inhibitory effect on the progress of isomerization at Asp1 and Asp7, respectively, by forming Asp-His complex that serves to slow down the conversion from Asp to isoAsp.