Intriguingly, EX1 kinetics usually reflect important transitions or intermediates in protein folding. For example, these patterns were seen when conditions caused a progressively larger Pimozide proportion of protein molecules to unfold, and also when proteins were allowed to refold after such a stress. If individual domains of a multi-domain protein have varying stabilities, peptides from each region exhibit bimodal mass peaks under specific, differing conditions. The presence of EX1 kinetics under non-denaturing conditions is extremely rare, but has been observed for a small set of SH3 domain proteins, including hematopoetic cell kinase and Lyn kinases. Our observation of bimodal deuteration spectra for multiple peptides comprising a specific monomer-monomer interface suggests that this specific region undergoes some degree of local ����unfolding����. Moreover, we believe that the cooccurrence of wild-type NPM1 granzyme B cleavage fragments corresponding to use of both D161 and D122 supports our DXMS analysis that wild-type NPM1 undergoes dynamic structural shifts, likely between two distinct conformations which favor stabilized Sodium ascorbate versus destabilized oligomers. However, it cannot be excluded that alternative initiation of translation occurred in wild-type NPM1 IVTT reactions, thus producing amino-terminally truncated constructs which contributed to the formation of SDS-stable oligomers and specific granzyme B cleavage fragments that match what were seen with M7-NPM IVTT reactions. Although our DXMS studies used conditions thought to reduce deuteration kinetics and destabilize NPM1 oligomers, we believe that interactions at the monomer-monomer interface, including the b-hairpin loop, are important for NPM1 conformations under physiological conditions. In support of this, we first defined the critical role of the b-hairpin loop by changing a key residue, thus generating a mutant, Y67E-NPM, which could not form SDS-stable oligomers, and furthermore, prevented wild-type NPM1 oligomer formation in a dominantnegative fashion.Second, we found that granzyme B cleavage of wild-type NPM1 in the presence of occurred at both D161 and D122, thereby producing fragment patterns which corresponded to granzyme B interactions with both stabilized and destabilized oligomers, as represented by M7-NPM and Y67E-NPM reaction products, respectively.