Symmetry of 4-Oxalocrotonate Tautomerase Trimers Influences Unfolding and Fragmentation in the Gas Phase

The recent discovery of asymmetric arrangements of trimers in the tautomerase superfamily (TSF) adds structural diversity to this already mechanistically diverse superfamily. Classification of asymmetric trimers has previously been determined using X-ray crystallography. Here, native mass spectrometry (MS) and ultraviolet photodissociation (UVPD) are employed as an integrated strategy for more rapid and sensitive differentiation of symmetric and asymmetric trimers. Specifically, the unfolding of symmetric and asymmetric trimers initiated by collisional heating was probed using UVPD, which revealed unique gas-phase unfolding pathways. Variations in UVPD patterns from native-like, compact trimeric structures to unfolded, extended conformations indicate a rearrangement of higher-order structure in the asymmetric trimers that are believed to be stabilized by salt-bridge triads, which are absent from the symmetric trimers. Consequently, the symmetric trimers were found to be less stable in the gas phase, resulting in enhanced UVPD fragmentation overall and a notable difference in higher-order re-structuring based on the extent of hydrogen migration of protein fragments. The increased stability of the asymmetric trimers may justify their evolution and concomitant diversification of the TSF. Facilitating the classification of TSF members as symmetric or asymmetric trimers assists in delineating the evolutionary history of the TSF.