Wieden, Hans-Joachim

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Browsing Wieden, Hans-Joachim by Author "De Laurentiis, Evelina I."

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    The C-terminal helix of Pseudomonas aeruginosa elongation factor Ts tunes EFT-Tu dynamics to modulate nucleotide exchange
    (American Society for Biochemistry and Molecular Biology, 2015) De Laurentiis, Evelina I.; Mercier, Evan; Wieden, Hans-Joachim
    Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli. Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu·EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts.
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    Identification of two structural elements important for ribosome-dependent GTPase activity of elongation factor 4 (EF4/LepA)
    (Nature Research, 2015) De Laurentiis, Evelina I.; Wieden, Hans-Joachim
    The bacterial translational GTPase EF4/LepA is structurally similar to the canonical elongation factor EF-G. While sharing core structural features with other translational GTPases, the function of EF4 remains unknown. Recent structural data locates the unique C-terminal domain (CTD) of EF4 in proximity to the ribosomal peptidyl transferase center (PTC). To investigate the functional role of EF4’s CTD we have constructed three C-terminal truncation variants.These variants are fully functional with respect to binding mant-GTP and mant-GDP as determined by rapid kinetics, as well as their intrinsic multiple turnover GTPase activity. Furthermore, they are able to form stable complexes with the 70S ribosome and 50S/30S ribosomal sub units.However,successive removal of the C-terminus impairs ribosome-dependent multiple turnover GTPase activity of EF4, which for the full-length protein is very similar to EF-G. Our findings suggest that the last 44 C-terminal amino acids of EF4 form a sub-domain within the C-terminal domain that is important for GTP-dependent function on the ribosome. Additionally, we show that efficient nucleotide hydrolysis by EF4 on the ribosome depends on a conserved histidine (His 81), similar to EF-G and EF-Tu.
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    Using rapid kinetics and molecular dynamics simulations to study biomolecular information processing and design
    (Biomath Forum, 2016) De Laurentiis, Evelina I.; Girodat, Dylan J.; Mercier, Evan; Wieden, Hans-Joachim
    Translation, the ribosome dependent synthesis of proteins, is the last step of gene expression. It is targeted by a large number of antibiotics that modulate, in one way or the other, the dynamic properties of the involved biomolecules. Protein synthesis is a highly conserved multi-step process facilitated by a large ribonucleoprotein complex (the ribosome) acting as a biomolecular assembler, converting genetic information provided as RNA transcripts (mRNA) into proteins. This process occurs with high speed and incredible accuracy within any living system (1). As such, accuracy is the key to maintaining the integrity of the genetic information to be reflected in the resulting proteins. The underlying design principles that enable the translation machinery to achieve these performance characteristics is of great interest for a large number of fields and applications, naming antibiotics design and the rational engineering of biomolecular machines just as two examples.