Design for Solubility May Reveal Induction of Amide Hydrogen/Deuterium Exchange by Protein Self-Association

[Display omitted] •Adnectin scaffold solubility is improved by combined bioinformatic and CamSol analysis.•Adnectins differing at a single amino acid position vary greatly in solubility.•Adnectin self-association is correlated with unexpected local increases in rates of HDX.•Fast HDX in just one strand of a native hairpin may be a hallmark of Adnectin associations.•Amide HDX may identify self-association with high resolution and sensitivity. Structural heterogeneity often constrains the characterization of aggregating proteins to indirect or low-resolution methods, obscuring mechanistic details of association. Here, we report progress in understanding the aggregation of Adnectins, engineered binding proteins with an immunoglobulin-like fold. We rationally design Adnectin solubility and measure amide hydrogen/deuterium exchange (HDX) under conditions that permit transient protein self-association. Protein–protein binding commonly slows rates of HDX; in contrast, we find that Adnectin association may induce faster HDX for certain amides, particularly in the C-terminal β-strand. In aggregation-prone proteins, we identify a pattern of very different rates of amide HDX for residues linked by reciprocal hydrogen bonds in the native structure. These results may be explained by local loss of native structure and formation of an inter-protein interface. Amide HDX induced by self-association, detected here by deliberate modulation of propensity for such interactions, may be a general phenomenon with the potential to expose mechanisms of aggregation by diverse proteins.