Through the creation of de novo miniproteins that bind to specific sites on other natural proteins, computational protein design is enabling a revolution in how biological systems can be manipulated for therapeutic and research applications. We have previously used temperature-dependent nuclear magnetic resonance (NMR) and replica exchange molecular dynamics simulations to explore the structural and chemical heterogeneity of an ultra-stable model miniprotein, EHEEᵣd2₀005. It has been shown to undergo rapid deamidation of asparagine 31 at high temperature to (iso) aspartate. Here, we further characterized this spontaneous modification using a novel computational approach to elucidate modification kinetics in real time using unlabeled 1D protein NMR. We have used 2D NMR experiments to validate the 1D NMR results and understand the deamidation products in greater detail, particularly focusing on the little understood isoaspartate product. Chemical shift perturbations for the isoaspartate product generally have the same sign but larger magnitude than the aspartate product, highlighting how lengthening the backbone by one bond produces outsized structural changes. This structural change causes the isoaspartate variant to be much less resistant to thermal denaturation. We find that the particularly deleterious isoaspartate product is more favored kinetically than thermodynamically, resulting in a transient high initial population. Our results highlight the power of NMR to identify and characterize protein isomers invisible to other techniques. Prospectively avoiding such modifications will be critical for the development of miniproteins into effective therapeutics.
Blejec et al. (Sun,) studied this question.