Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances

Abstract

Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections, and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary-structure, protein hydrodynamics and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance. Namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites.

Identification of chlorinated products from tyrosine and tyrosyl dipeptides during chlorination: a computational study

“…First, as simplied model reaction products of chlorine with proteins, they modify the protein structure and disrupt their functions relevant to pathogen inactivation. 1,2 Second, as an emerging class of disinfection byproducts (DBPs), they exhibit even higher cytoand genotoxicity than the currently regulated DBPs, [3][4][5] and their decomposition products also have odor problems and potential health risks to humans. [6][7][8] Moreover, proteins, peptides, and amino acids, as the precursors of chlorinated amino acids and peptides, constitute the majority of not only the organic nitrogen in cell mass 9 but also the dissolved organic nitrogen (DON) in water.…”

Lysine and Arginine Reactivity and Transformation Products during Peptide Chlorination

“…Thus, within ∼0.5 h of chlorine contact, the cationic side chains of lysine and arginine decorating protein exteriors are transformed into neutral functional groups. This conversion fundamentally alters interactions with neighboring amino acids and thereby could contribute to the loss of the protein structure and enzymatic activity observed during protein chlorination ,, and ultimately pathogen inactivation . The conversion should also enhance protein hydrophobicity, contributing to the observed increase in their tendency to adhere to hydrophobic surfaces after chlorination .…”

Evaluation of Histidine Reactivity and Byproduct Formation during Peptide Chlorination

“…Chlorine reactions with the viral capsid proteins in MS2 bacteriophage accounted for ∼45% of the MS2 inactivation . The covalent modifications to amino acid side chains alter their interactions within proteins, disrupting protein structure and function. , The dissolved organic matter (DOM) serving as the precursor for DBPs in drinking water supplies ultimately derives from biomolecules. Recent DBP research has focused on nitrogen-based DBPs (N-DBPs) as important contributors to the DBP-associated toxicity of disinfected waters, based upon their higher toxic potency-weighted concentrations relative to carbon-based DBPs, including regulated trihalomethanes (THMs) and haloacetic acids. − Among biomolecules contributing to DOM, protein is an important N-DBP precursor, since protein accounts for the majority of organic nitrogen in cell mass (e.g., ∼55% in Escherichia coli ).…”

“…The covalent modifications to amino acids resulting from chlorination alter their interactions within proteins, contributing to the loss of structure and function, , and pathogen inactivation. These same transformation products are disinfection byproducts.…”

“…1 The covalent modifications to amino acid side chains alter their interactions within proteins, disrupting protein structure and function. 2,3 The dissolved organic matter (DOM) serving as the precursor for DBPs in drinking water supplies ultimately derives from biomolecules. Recent DBP research has focused on nitrogen-based DBPs (N-DBPs) as important contributors to the DBP-associated toxicity of disinfected waters, based upon their higher toxic potencyweighted concentrations relative to carbon-based DBPs, including regulated trihalomethanes (THMs) and haloacetic acids.…”