| Zusammenfassung: | The nature of biological radiation resistance is a question that has fascinated and inspired researchers since the first discovery of extremely radiation resistant organisms like Deinococcus radiodurans. To date many tightly coordinated mechanisms have been identified that lead to the emergence of the phenotype of extreme radiation resistance but only in the last decade was it found that proteome ... The nature of biological radiation resistance is a question that has fascinated and inspired researchers since the first discovery of extremely radiation resistant organisms like Deinococcus radiodurans. To date many tightly coordinated mechanisms have been identified that lead to the emergence of the phenotype of extreme radiation resistance but only in the last decade was it found that proteome protection constitutes survival not DNA protection. This thesis aims to provide a molecular understanding of how protein sequence and structure evolved to mitigate the damaging effects of ionising radiation in the environment. To systematically study whether the amino acid composition (primary sequence) is the source of a protein radiation resistance free amino acids, specifically tryptophan, has been soaked into multiple protein crystals and the average response to radiation insult has been compared with their apo counterparts. By collecting dose series measurements and using online UV/Vis spectroscopy the effect on global and specific damage rates has been analysed, showing that for lysozyme, thaumatin, AcNiR and 4HB1 no significant radio protective effect could be achieved by supplementing a protein crystal with tryptophan. Interestingly, one 4HB1 crystal that had been soaked with 100 mM tryptophan survived a total absorbed dose of 182.3 MGy (half dose of 64.43 MGy). Although the exact conditions leading to this result were not reproducible, this result constitutes an unprecedented case of extreme radiation tolerance in an intense X-ray beam and was hence included in the analysis.
A bioinformatics study has been performed, which analysed the amino acid composition radiation hard bacteria and compared them with 8000 bacteria proteomes. The amino acid distribution of radiation resistant organisms showed no common bias towards a particular amino acid or combination of amino acids. Clustering a subset of 200 proteomes from each domain showed that the phylogenetic filiation can be predicted from the amino acid composition, the phenomenon of extreme radiation resistance however can not be predicted. This result supports the conclusion that there is no single amino acid or combination thereof that are the source of a proteins radiation resistance.
Oxidative modifications are not uncommon in cysteine proteases and have been shown affect and even inhibit enzymatic activity. Recent structures of the SARS-CoV-2 main protease (Mpro) show indications for similar modifications despite the fact that the enzyme is naturally expressed in the cytosol which is considered to be of reducing nature. To determine whether this modification is an artefact of the purification strategy and what impact such a modification on the enzyme activity as well as recent active-site drug screening efforts would have, Mpro was purified under aerobic conditions (as reported by most studies), aerobic conditions without the use of reducing agents and anaerobic conditions. X-ray diffraction data of Mpro from both aerobic purifications indicate oxidative modifications of the active site Cys145. Using mass spectrometry, we could show that in the presence of reducing agents Mpro is only oxidised when the effectiveness of reducing agents decays, e.g. during long crystallisation periods but not during the purification itself. Without reducing agents at latest after 12 days Mpro molecules can be expected to contain sulfenic acid (-SO) and sulfinic acid (-SO2) modifications at the active site Cys145. As a result the oxidised enzyme has a specificity constant approximately 50 % lower than unmodified Mpro for the substrate Ac-Abu-Tle-Leu-Gln-AMC. By purifying and crystallising Mpro under anaerobic conditions this study shows that the oxidation of the enzyme can be avoided and is therefore likely an artefact of the in vitro enzyme processing. |
