Giant Carbon Nano-Test Tubes as Versatile Imaging Vessels for High-Resolution and In Situ Observation of Proteins.

Cryogenic electron microscopy is one of the fastest and most robust methods for capturing high-resolution images of proteins, but stringent sample preparation, imaging conditions, and in situ radiation damage inflicted during data acquisition directly affect the resolution and ability to capture dynamic details, thereby limiting its broader utilization and adoption for protein studies. We addressed these drawbacks by introducing synthesized giant carbon nano-test tubes (GCNTTs) as radiation-insulating materials that lessen the irradiation impact on the protein during data acquisition, physical molecular concentrators that localize the proteins within a nanoscale field of view, and vessels that create a microenvironment for solution-phase imaging. High-resolution electron microscopy images of single and aggregated hemoglobin molecules within GCNTTs in both solid and solution states were acquired. Subsequent scanning transmission electron microscopy, small-angle neutron scattering, and fluorescence studies demonstrated that the GCNTT vessel protected the hemoglobin molecules from electron irradiation-, light-, or heat-induced denaturation. To demonstrate the robustness of GCNTT as an imaging platform that could potentially augment the study of proteins, we demonstrated the robustness of the GCNTT technique to image an alternative protein, d-fructose dehydrogenase, after cyclic voltammetry experiments to review encapsulation and binding insights. Given the simplicity of the material synthesis, sample preparation, and imaging technique, GCNTT is a promising imaging companion for high-resolution, single, and dynamic protein studies under electron microscopy.

Successful Mesoporous Silica Encapsulation of Optimally Functional EcDOS (E. coli Direct Oxygen Sensor), a Heme-based O2-Sensing Phosphodiesterase.

The heme-based O2 sensor from Escherichia coli, EcDOS, exerts phosphodiesterase activity towards cyclic-di-GMP (c-di-GMP), an important second messenger that regulates biofilm formation, virulence, and other important functions necessary for bacterial survival. EcDOS is a two-domain protein composed of an N-terminal heme-bound O2-sensing domain and a C-terminal functional domain. O2 binding to the heme Fe(II) complex in the O2-sensing domain substantially enhances the catalytic activity of the functional domain, a property with potentially promising medical applications. Mesoporous silica is a useful material with finite-state machine-like features suitable for mediating numerous enzymatic functions. Here, we successfully encapsulated EcDOS into mesoporous silica, and demonstrated that encapsulated EcDOS was substantially activated by CO, an alternative signaling molecule used in place of O2, exhibiting the same activity as the native enzyme in aqueous solution. Encapsulated EcDOS was sufficiently stable to exert its enzymatic function over several experimental cycles under aerobic conditions at room temperature. Thus, the present study demonstrates the successful encapsulation of the heme-based O2 sensor EcDOS into mesoporous silica and shows that the native gas-stimulated function of EcDOS is well conserved. As such, this represents the first application of mesoporous silica to an oxygen-sensing-or any gas-sensing-enzyme.

An enzyme-encapsulated microreactor for efficient theanine synthesis.

A flow-type microreactor containing glutaminase-mesoporous silica composites with 10.6 nm pore diameter (TMPS10.6) was developed for the continuous synthesis of theanine, a unique amino acid. High enzymatic activity was exhibited by the local control of the reaction temperature.

Enhancement in thermal stability and resistance to denaturants of lipase encapsulated in mesoporous silica with alkyltrimethylammonium (CTAB).

We assembled a highly durable conjugate with both a high-density accumulation and a regular array of lipase, by encapsulating it in mesoporous silica (FSM) with alkyltrimethylammonium (CTAB) chains on the surface. The activity for hydrolyzing esters of the lipase immobilized in mesoporous silica was linearly related to the concentration of lipase, whereas that of non-immobilized lipase showed saturation due to self-aggregation at a high concentration. The lipase conjugate also had increased resistance to heating when stayed in the silica coupling with CTAB. In addition, encapsulating the enzyme with FSM coupled CTAB caused the lipase to remain stable even in the presence of urea and trypsin, suggesting that the encapsulation prevented dissociation and denaturing. This conjugate had much higher activity and much higher stability for hydrolyzing esters when compared to the native lipase. These results show that FSM provides suitable support for the immobilization and dispersion of proteins in mesopores with disintegration of the aggregates.