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

Abstract

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.

Expanding Orbitrap Collision Cross-Section Measurements to Native Protein Applications Through Kinetic Energy and Signal Decay Analysis

“…The resulting CIU curve is shown in Figure , and the CCS increases by 25% during the unfolding process prior to reaching a plateau around 2858 Å 2 . The CIU curve is similar to ones measured by ion mobility, in which the increase in CCS during CIU frequently ranges from 30 to 50%. − …”

Expanding Orbitrap collision cross section measurements to native protein applications through kinetic energy and signal decay analysis

“…The CIU curve is similar to ones measured by ion mobility, in which the increase in CCS during CIU frequently ranges from 30-50%. [56][57][58][59][60] Figure 5. Orbitrap CCS of native-like carbonic anhydrase (9+, 29 kDa monomer, m/z 3233) relative to the collision energy used for in-source CID to cause collisional heating.…”

Ultraviolet Photodissociation Reveals the Molecular Mechanism of Crown Ether Microsolvation Effect on the Gas-Phase Native-like Protein Structure

“…The coverages of sequence cleavage of Mb ions with charge states 6+, 7+, 8+, and 9+ were 36.8%, 85.8%, 90.7%, and 91.0%, respectively (Figures A and S3–S6). The stepwise increases in charge states of the a and a+1 fragment ions in 193 nm UVPD could be utilized to localize the proton (positive charge) along the protein sequence, which was termed as charge site analysis. , The sharp alterations of the charge state distributions of a and a+1 fragments indicated the positions of proton localization, while the tailing distribution of charge states could be attributed to the multiple protonation forms in corresponding regions (Figure B). For example, the sharp change of a16 + to a17 2+ indicated the protonation at Val17 for Mb 9+ .…”