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Effects of atmospheric pressure plasmas on proteins are studied to assess the quality of plasma decontamination and to gain insights into plasma-triggered molecular events underlying observations made in plasma medicine on the cellular, organ, and systemic level. Atmospheric pressure plasma treatment has been reported to cause protein degradation. Degradation products, however, have not been characterized. Treating different model proteins in aqueous solution with a DBD plasma, we confirmed with different methods (Bradford assay, application of Lambert–Beer’s law on absorption measurements at 280 nm, ninhydrin assay, size exclusion chromatography, SDS-PAGE) that protein degradation takes place. Peptides of different sizes were detected by size exclusion chromatography. The ninhydrin assay indicated that peptide bonds are cleaved. In the presence of hydroxyl radical scavenger d-mannitol, the concentration of amino termini formed during the initial 10 min of plasma treatment was reduced by 96%, while at longer treatment times mannitol did no longer prevent the formation of amino termini, indicating that hydroxyl radicals play an important role in the initial cleaving of peptide bonds in the protein, but other mechanisms are at play in cleaving the peptide bonds in the resulting peptides. The generation of peptides has implications for plasma decontamination and plasma medicine. It is critical to verify that plasma decontamination processes do not result in protein fragments with undesired properties. In plasma medicine, plasma-generated protein fragments may act as molecular triggers in treated cells, tissues, or patients, e.g., regulating signaling cascades in a protease-like fashion.

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidA’s chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA’s chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N- chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCl’s chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

A molecular���level understanding of the effects of atmospheric���pressure plasma on biological samples requires knowledge of the effects on proteins. Superoxide dismutases, which detoxify superoxide under oxidative stress conditions, play akey role in bacterial plasma resistance. Investigation of the impact of dielectric barrier discharge (DBD) treatment on purified superoxide dismutases SodA and SodB of Escherichia coli showed that DBD treatment caused a rapid protein degradation, with only 8% of protein remaining after 10 min. The affinity of SodA for the metal cofactor Mn2+was reduced. Mass spectrometry, in conjunction with coupled���cluster calculations, revealed that modifications of amino acid residues in the active site can explain the decreased metal affinity and a distortion ofthe coordination geometry responsible for the activity loss. sponsible for the activity loss., publishedVersion

Non-equilibrium atmospheric-pressure plasmas are an alternative means to sterilize and disinfect. Plasma-mediated protein aggregation has been identified as one of the mechanisms responsible for the antibacterial features of plasma. Heat shock protein 33 (Hsp33) is a chaperone with holdase function that is activated when oxidative stress and unfolding conditions coincide. In its active form, it binds unfolded proteins and prevents their aggregation. Here we analyse the influence of plasma on the structure and function of Hsp33 of Escherichia coli using a dielectric barrier discharge plasma. While most other proteins studied so far were rapidly inactivated by atmospheric-pressure plasma, exposure to plasma activated Hsp33. Both, oxidation of cysteine residues and partial unfolding of Hsp33 were observed after plasma treatment. Plasma-mediated activation of Hsp33 was reversible by reducing agents, indicating that cysteine residues critical for regulation of Hsp33 activity were not irreversibly oxidized. However, the reduction yielded a protein that did not regain its original fold. Nevertheless, a second round of plasma treatment resulted again in a fully active protein that was unfolded to an even higher degree. These conformational states were not previously observed after chemical activation with HOCl. Thus, although we could detect the formation of HOCl in the liquid phase during plasma treatment, we conclude that other species must be involved in plasma activation of Hsp33. E. coli cells over-expressing the Hsp33-encoding gene hslO from a plasmid showed increased survival rates when treated with plasma while an hslO deletion mutant was hypersensitive emphasizing the importance of protein aggregation as an inactivation mechanism of plasma.

Non-thermal atmospheric pressure plasmas are investigated as augmenting therapy to combat bacterial infections. The strong antibacterial effects of plasmas are attributed to the complex mixture of reactive species, (V)UV radiation and electric fields. The experience with antibiotics is that upon their introduction as medicines, resistance occurs in pathogens and spreads. To assess the possibility of bacterial resistance developing against plasma, we investigated intrinsic protective mechanisms that allow Escherichia coli to survive plasma stress. We performed a genome-wide screening of single-gene knockout mutants of E. coli and identified 87 mutants that are hypersensitive to the effluent of a microscale atmospheric pressure plasma jet. For selected genes ( cysB , mntH , rep and iscS ) we showed in complementation studies that plasma resistance can be restored and increased above wild-type levels upon over-expression. To identify plasma-derived components that the 87 genes confer resistance against, mutants were tested for hypersensitivity against individual stressors (hydrogen peroxide, superoxide, hydroxyl radicals, ozone, HOCl, peroxynitrite, NO•, nitrite, nitrate, HNO 3 , acid stress, diamide, heat stress and detergents). k-means++ clustering revealed that most genes protect from hydrogen peroxide, superoxide and/or nitric oxide. In conclusion, individual bacterial genes confer resistance against plasma providing insights into the antibacterial mechanisms of plasma.

In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7 T (Acd DPN7 ) during degradation of 3,3′-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). Acd DPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. Acd S110 , Acd B4 , Acd H16 , and Acd KT2440 were able to dehydrogenate isobutyryl-CoA. Acd KT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas Acd DSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only Acd TBEA6 , Acd N-1 , and Acd LB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for Acd BL21 and Acd WT001 . Spectrophotometric assays provided apparent K m and V max values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of uncharacterized acyl-CoA dehydrogenases. Furthermore, C. necator N-1 was found to utilize 3SP as the sole source of carbon and energy.