Assembly and function of the tRNA-modifying GTPase MnmE adsorbed to surface functionalized bioactive glass.
Protein adsorption onto solid surfaces is a common phenomenon in tissue engineering related applications, and considerable progress was achieved in this field. However, there are still unanswered questions or contradictory opinions concerning details of the protein's structure, conformational changes, or aggregation once adsorbed onto solid surfaces. Electron paramagnetic resonance (EPR) spectroscopy and site-directed spin labeling (SDSL) were employed in this work to investigate the conformational changes and dynamics of the tRNA-modifying dimeric protein MnmE from E. coli, an ortholog of the human GTPBP3, upon adsorption on bioactive glass mimicking the composition of the classical 45S5 Bioglass. In addition, prior to protein attachment, the bioactive glass surface was modified with the protein coupling agent glutaraldehyde. Continuous wave EPR spectra of different spin labeled MnmE mutants were recorded to assess the dynamics of the attached spin labels before and after protein adsorption. The area of the continuous wave (cw)-EPR absorption spectrum was further used to determine the amount of the attached protein. Double electron-electron resonance (DEER) experiments were conducted to measure distances between the spin labels before and after adsorption. The results revealed that the contact regions between MnmE and the bioactive glass surface are located at the G domains and at the N-terminal domains. The low modulation depths of all DEER time traces recorded for the adsorbed single MnmE mutants, corroborated with the DEER measurements performed on MnmE double mutants, show that the adsorption process leads to dissociation of the dimer and alters the tertiary structure of MnmE, thereby abolishing its functionality. However, glutaraldehyde reduces the aggressiveness of the adsorption process and improves the stability of the protein attachment.
Faculty of Physics and Institute of Interdisciplinary Research in Bio-Nano-Sciences, Babes-Bolyai University , Cluj-Napoca, 400084, Romania.
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Gruian
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Boehme
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Simon
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HJ
Steinhoff
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JP
Klare
D000327:Adsorption; D002516:Ceramics / Q000737:chemistry*; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005898:Glass / Q000737:chemistry*; D009154:Mutation; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism; D013113:Spin Labels
D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D013113:Spin Labels; C425330:bioactive glass 45S5; D012343:RNA, Transfer; D020558:GTP Phosphohydrolases