Assembly and Function of the tRNA-Modifying GTPase MnmE Adsorbed to Surface Functionalized Bioactive Glass C. Gruian, †,‡ S. Boehme, ‡ S. Simon, † H.-J. Steinhoff, ‡ and J. P. Klare* ,‡ † Faculty of Physics and Institute of Interdisciplinary Research in Bio-Nano-Sciences, Babes-Bolyai University, Cluj-Napoca, 400084, Romania ‡ Department of Physics, University of Osnabrü ck, Osnabrü ck, 49069, Germany * S Supporting Information ABSTRACT: Protein adsorption onto solid surfaces is a common phenomenon in tissue engineering related applica- tions, 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. KEYWORDS: bioglass, MnmE, protein adsorption, glutaraldehyde, biocompatibility, site-directed spin labeling, EPR spectroscopy ■ INTRODUCTION The biological functions of proteins, enzymes, and other biological molecules are associated with structural dynamics and are often realized by changes in conformation. A major interest in biology is to understand the function of biomacromolecules, in order to establish how a biological process proceeds from simple structural changes in biomole- cules to the final and often complex biological function. In this respect, information concerning kinetics, structure, and conformational dynamics of biomacromolecules is of primary importance. Besides the information about their behavior in the native environment, for optimal performance in biomedical or physiological applications, it is important to investigate how complex biomolecules change their structure when interacting with foreign materials and surfaces. For example, adsorption of protein molecules onto solid surfaces plays a key role in many natural processes and frequently results in conformational and orientation changes within the adsorbed layer. 1,2 An intensive knowledge of the protein adsorption process is not only beneficial for optimization of the surface structure of biomaterials but also helpful to develop specific applications within the field of biomedicine. 3 In particular, protein adsorption on osteoinductive bioceramic type surfaces was extensively studied in the past years since it plays a vital role during bone tissue regeneration 4,5 and helps in understanding the mechanisms of bioactivity. 6-8 Several experimental and theoretical approaches recently reported that adsorbed proteins can also influence surface mineralization of the substrate by affecting the nucleation and growth as well as the morphology, size, and orientation of Ca-P crystals. 9-11 Proteins are spontaneously adsorbed onto bioactive glass (BG), long before Ca-P precipitation; actually, the first processes that occur at the BG surface upon immersion in simulated body fluid (SBF) are ion release (dissolution) and SBF diffusion. 12,13 Notwith- standing, details concerning protein structure and dynamics after adsorption, the exact amount of the protein attached to Received: February 13, 2014 Accepted: May 1, 2014 Published: May 1, 2014 Research Article www.acsami.org © 2014 American Chemical Society 7615 dx.doi.org/10.1021/am500933e | ACS Appl. Mater. Interfaces 2014, 6, 7615-7625