Co-translational processing and membrane insertion of proteins in bacteria
by Lena A. K. Bögeholz
Date of Examination:2022-09-13
Date of issue:2022-11-04
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Kai Tittmann
Referee:Dr. Alexander Stein
Referee:Prof. Dr. Wolfgang Wintermeyer
Referee:Dr. Alex Faesen
Referee:Prof. Dr. Hauke Hillen
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EnglishProtein biogenesis entails the synthesis of polypeptides on the ribosome, their co- and post-translational maturation, folding, and, in the case of membrane proteins, insertion into the lipid bilayer. Bacterial proteins are synthesized with a formyl group on the N-terminus and the first processing step of the nascent chain is the removal of this formyl group by peptide deformylase (PDF). The majority of new proteins undergoes deformylation which is essential in bacteria and a requirement for the subsequent processing step. Here, we analyzed the kinetic mechanism of deformylation using ribosome nascent chain complexes (RNCs) and actively translating ribosomes as substrates. Binding and dissociation of PDF and RNC occur rapidly, allowing for efficient scanning of formylated substrates in the cell. Rapid hydrolysis of the formyl group stops scanning and is followed by the rate-limiting conformational rearrangement of the nascent chain. Therefore, release of the RNC by PDF is slow, which limits further N-terminal processing and may affect folding of nascent proteins. PDF is a promising drug target for antimicrobials, which prompted us to test whether deformylation can be prevented by novel analogues of the natural PDF inhibitor fumimycin, however, the analogues had only small effects. During the biogenesis of inner membrane proteins, deformylation is inhibited by the signal recognition particle which ensures that the transmembrane helices (TMs) of the nascent membrane protein are co-translationally inserted into the lipid bilayer where the protein begins to fold. Insertion occurs with the help of the SecYEG translocon in bacteria but how TMs move into the membrane is not well understood. To monitor the co-translational insertion of TM1 and TM2 of the model protein EmrD, we performed real-time FRET experiments with fluorescence labels on the nascent chain and the translocon. We show that TM1 and TM2 are in close proximity to each other while partitioning into the membrane and that the two TMS remain close to the translocon afterwards. While the N-terminus resides in a relatively stable position, TM2 is more dynamic and undergoes conformational rearrangements that indicate the onset of protein folding before EmrD has been fully synthesized.
Keywords: N-terminal processing; Membrane protein folding; Co-translational protein folding