Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility (SA) have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the E. coli proteome using several proteomic methodologies and globally measured oxidation rates of methionines in the presence and absence of tertiary structure, as well as folding stabilities of methionine containing domains. The data indicate that buried methionines have a wide range of protection factors against oxidation which correlate strongly with folding stabilities. Concordantly, we show that in comparison to E. coli, the proteome of the thermophile T. thermophilus is significantly more stable and thus more resistant to methionine oxidation. These results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and suggest a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures.

Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods

Plasma fibrinogen is an important coagulation factor and susceptible to post-translational modification by oxidants. We have reported impairment of fibrin polymerization after exposure to hypochlorous acid (HOCl) and increased methionine oxidation of fibrinogen in severely injured trauma patients. Molecular dynamics suggests that methionine oxidation poses a mechanistic link between oxidative stress and coagulation through protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We utilized polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to investigate the hypothesis that HOCl oxidation alters fibrinogen conformation and T2 relaxation time of water protons in the fibrin gels. We have demonstrated that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma fibrinogen solution disrupted lateral aggregation of protofibrils similarly to competitive inhibition of fibrin polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of fibrinogen protons demonstrated that the diffusion coefficient of fibrinogen increased by 17.4%, suggesting the oxidized fibrinogen was more compact and fast motion in the prefibrillar state. 2D-NMR analysis reflected that water protons existed as bulk water (T2) and intermediate water (T2i) in the control plasma fibrin. Bulk water T2 relaxation time was increased twofold and correlated positively with the level of HOCl oxidation. However, T2 relaxation of the oxidized plasma fibrin gels was dominated by intermediate water. Oxidation induced thinner fibers, in which less water is released into the bulk and water fraction in the hydration shell was increased. We have confirmed that T2 relaxation is affected by the self-assembly of fibers and stiffness of the plasma fibrin gel. We propose that water protons can serve as an NMR signature to probe oxidative rearrangement of the fibrin clot.

MICAL2 fine-tunes Arp2/3 for actin branching

The ARP2/3 complex promotes branched actin networks, but the importance of specific subunit isoforms is unclear. In this issue, Galloni, Carra, et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202102043) show that MICAL2 mediates methionine oxidation of ARP3B, thus destabilizing ARP2/3 complexes and leading to disassembly of branched actin filaments.

Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods

Plasma fibrinogen is an important coagulation factor that is susceptible to post-translational modification by oxidants. We have reported altered fibrin polymerization and increased methionine oxidation in fibrinogen after exposure to hypochlorous acid (HOCl), and similarly in the fibrinogen of severely injured trauma patients. Molecular dynamics suggests that methionine oxidation offers a mechanistic link between oxidative stress and coagulation through fibrin protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We used polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to test the hypothesis that HOCl oxidation alters fibrinogen conformation in the prefibrillar state and T2 water surface relaxation of fibrin fiber assemblies. We found that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma disrupted fibrin polymerization similarly to competitive inhibition of polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of 1H fibrinogen at 25oC demonstrated that fibrinogen oxidation increased translational diffusion coefficient by 17.4%, suggesting a more compact and rapidly translational motion of the protein with oxidation. 2D-NMR analysis of control plasma fibrin gels indicated that water existed in two states, namely intermediate (T2i) in the hydration shell of fibrin fibers, and bulk (T2) within the gel. T2 relaxation of bulk water protons was decreased 2-fold in oxidized fibrin gels and was inversely proportional to gel fiber density (T2). The fast exchange of water protons between hydration shell (T2i) and bulk water, indicating oxidation increased fiber hydration and formed densely packed fibrin gels. We have confirmed experimentally that HOCl oxidation affected native fibrinogen and fibrin gel structures and have demonstrated that NMR can serve as a valuable tool to probe the oxidative rearrangement of fibrin clot structure.