Assembly and Function of the tRNA-Modifying GTPase MnmE Adsorbed to Surface Functionalized Bioactive Glass

Abstract

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.

Glass-Ceramics: Fundamental Aspects Regarding the Interaction with Proteins

“…7) [30]. Further information needed to confirm this assumption was obtained from pulse EPR experiments (DEER).…”

“…In the presence of GDP•AlF x , a transition state mimic for GTP hydrolysis, the G domains contact each other by overcoming a 20-30 Ǻ distance gap (Fig. 4) [30]. The multidomain architecture and the observed large conformational change occurring upon activation renders this protein an ideal model system to study the influence of protein-BG interaction on functional conformational dynamics.…”

“…The multidomain architecture and the observed large conformational change occurring upon activation renders this protein an ideal model system to study the influence of protein-BG interaction on functional conformational dynamics. The changes induced in the MnmE structure by the adsorption process were investigated by SDSL-EPR spectroscopy in two steps of its GTPase cycle: the apo-state (nucleotide free) and the GTP hydrolysis transition (GDP•AlF x bound) state [30].…”

“…The amino acids at four positions within MnmE were mutated to cysteine and subsequently spin labeled with the (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) methanethiosulfonate spin label (MTSSL) [30]: Ser278 and Glu287 (both located in the G-domain); Ile105 and Lys95 (both situated in the N terminal domain of the protein) (see Fig. 4).…”

“…3). The mobile component corresponds to the signal provided by spin labels that have less constrains in their surroundings and that are exposed to aqueous environment [30]. Upon adsorption on the bioactive glass, for both spin-labeled proteins the equilibrium between the two states is significantly shifted towards component I, indicating a conformational change (Figs.…”