Simulated and experimental force spectroscopy of lysozyme on silica.

The force spectra of proteins detaching from oxide surfaces measured by atomic force microscopy (AFM) often present complex patterns of peaks, which are difficult to correlate with individual bond-breaking events at the atomic scale. In this work we rationalize experimental AFM force spectra of hen-egg-white lysozyme detaching from silica by means of all-atom steered molecular dynamics (SMD) simulations. In particular, we demonstrate that the native tertiary structure of lysozyme is preserved if, and only if, its four intramolecular disulfide bridges are intact. Otherwise, the protein pulled off the surface undergoes severe unfolding, which is well captured by SMD simulations in explicit solvent. Implicit solvent simulations, on the contrary, wrongly predict protein unfolding even in the presence of S-S bridges, due to the lack of additional structural stabilization provided by the water's hydrogen-bond network within and surrounding the protein. On the basis of our combined experimental and theoretical findings, we infer that the rugged force spectra characteristic of lysozyme/silica interfaces are not due to the successive breaking of internal disulfide bonds leading to partial unfolding events. Rather, they reflect the detachment of several molecules bound to the same AFM tip, each anchored to the surface via multiple hydrogen and ionic bonds.

Atomistic Details of Chymotrypsin Conformational Changes upon Adsorption on Silica.

Adsorption of enzymes on solid surfaces may lead to conformational changes that reduce their catalytic conversion activity and are thus detrimental to the efficiency of biotechnology or biosensing applications. This work is a joint theoretical and experimental endeavor in which we identify and quantify the conformational changes that chymotrypsin undergoes when in contact with the surface of amorphous silica nanoparticles. For this purpose, we use circular dichroism spectroscopy, standard molecular dynamics, and advanced-sampling methods. Only the combination of these techniques allowed us to pinpoint a destabilization effect of silica on specific structural motifs of chymotrypsin. They are linked by the possibility of theoretically predicting CD spectra, allowing us to elucidate the source of the experimentally observed spectral changes. We find that chymotrypsin loses part of its helical content upon adsorption, with minor perturbation of its overall tertiary structure, associated with changes in the aromatic interactions. We demonstrate that the C-terminal helical fragment is unfolded as an isolated oligopeptide in pure water, folded as an α-helix as terminus of chymotrypsin in solution, and again partly disordered when the protein is adsorbed on silica. We believe that the joint methodology introduced in this manuscript has a direct general applicability to investigate any biomolecule-inorganic surface system. Methods to theoretically predict circular dichroism spectra from atomistic simulations were compared and improved. The drawbacks of the approaches are discussed; in particular, the limited capability of advanced-sampling MD schemes to explore the conformational phase space of large proteins and the dependency of the predicted ellipticity bands on the choice of calculation parameters.

Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials.

Physisorption of α-chymotrypsin on SiO2 and TiO2: A comparative study via experiments and molecular dynamics simulations.

In order to understand fundamental interactions at the interface between immobilized enzymes and ceramic supports, the authors compare the adsorption features of chymotrypsin on SiO2 and TiO2 colloidal particles by means of a combination of adsorption experiments and molecular dynamics simulations. While the dependency of the adsorption amount on pH is consistent with the trend predicted the Derjaguin-Landau-Verwey-Overbeek theory, other effects can only be rationalized if the atomic-scale details of the water-mediated protein-surface interactions are considered. On both surfaces, a clear driving force for the formation of a double monolayer at the saturation coverage is found. Although nearly equal free energies of adsorption are estimated on the two materials via a Langmuir adsorption analysis, about 50% more proteins per unit of surface can be accommodated on TiO2 than on SiO2. This is probably due to the lower surface diffusion mobility of the adsorbed protein in the latter case. Surface anchoring is realized by a combination of direct ionic interactions between charged proteins and surface sites (more pronounced for SiO2) and distinct structuring of the surface hydration layers in which the contact residues are embedded (more pronounced for TiO2). Finally, normalization of the data with respect to particle surface areas accessible to the proteins, rather than determined by means of the Brunauer-Emmett-Teller nitrogen adsorption isotherm, is crucial for a correct interpretation of the results.

Physisorption of enzymatically active chymotrypsin on titania colloidal particles.

In this study we use a straightforward experimental method to probe the presence and activity of the proteolytic enzyme α-chymotrypsin adsorbed on titania colloidal particles. We show that the adsorption of α-chymotrypsin on the particles is irreversible and pH-dependent. At pH 8 the amount of adsorbed chymotrypsin is threefold higher compared to the adsorption at pH 5. However, we observe that the adsorption is accompanied by a substantial loss of enzymatic activity, and only around 6-9% of the initial enzyme activity is retained. A Michaelis-Menten kinetics analysis of both unbound and TiO2-bound chymotrypsin shows that the K(M) value is increased from ∼10 µM for free chymotrypsin to ∼40 µM for the particle bound enzyme. Such activity decrease could be related by the hindered accessibility of substrate to the active site of adsorbed chymotrypsin, or by adsorption-induced structural changes. Our simple experimental method does not require any complex technical equipment, can be applied to a broad range of hydrolytic enzymes and to various types of colloidal materials. Our approach allows an easy, fast and reliable determination of particle surface-bound enzyme activity and has high potential for development of future enzyme-based biotechnological and industrial processes.