Jecmen T. et al.: Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium (Int J Mol Sci, 2023, Jul 22;24(14):11779, doi: 10.3390/ijms241411779) Jecmen T. et al.: Photo-initiated crosslinking extends mapping of the protein-protein interface to membrane-embedded portions of cytochromes P450 2B4 and b5 (Methods, 2015, Nov 1;89:128-37, doi: 10.1016/j.ymeth.2015.07.015)
Preliminary scope of work
V práci budou exprimovány vybrané mutantní cytochromy b5 v auxotrofních bakteriích se zablokovanou drahou syntézy methioninu, které umožňují vnesení nepřirozeného aminokyselinového analogu (foto-methioninu, azidohomoalaninu nebo homopropargylglycinu) do sekvence proteinu. V proteinech bude určeno procento substituce methioninu jeho analogem pomocí hmotnostní spektrometrie a následně budou purifikovány. Čisté preparáty budou využity ke studii interakcí a elektronového přenosu mezi cytochromy b5 a P450.
Preliminary scope of work in English
Selected mutant cytochromes b5 will be expressed in auxotrophic bacteria with blocked methionine synthesis pathway, which allow the introduction of an unnatural amino acid analogue (photo-methionine, azidohomoalanine or homopropargylglycine) into the protein sequence. The percentage of methionine substitution by its analogue in the proteins will be determined by mass spectrometry and subsequently purified. The purified preparations will be used to study interactions and electron transfer between cytochromes b5 and P450.
