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In this study, we report a detailed experimental, binding free energy calculation and molecular dynamics (MD) simulation investigation of the interactions of carboxylic-functionalized multi-walled carbon nanotubes (COOH-f-MWCNTs) with porcine trypsin (pTry). The enzyme exhibits decreased thermostability at 330K in the presence of COOH-f-MWCNTs. Furthermore, the activity of pTry also decreases in the presence of COOH-f-MWCNTs. The restricted diffusion of the substrate to the active site of the enzyme was observed in the experiment. The MD simulation analysis suggested that this could be because of the blocking of the S1 pocket of pTry, which plays a vital role in the substrate selectivity. The intrinsic fluorescence of pTry is quenched with increase in the COOH-f-MWCNTs concentration. Circular dichroism (CD) and UV-visible absorption spectroscopies indicate the ability of COOH-f-MWCNTs to experience conformational change in the native structure of the enzyme. The binding free energy calculations also show that electrostatics, π-cation, and π-π stacking interactions play important roles in the binding of the carboxylated CNTs with pTry. The MD simulation results demonstrated that the carboxylated CNTs adsorb to the enzyme stronger than the CNT without the-COOH groups. Our observations can provide an example of the nanoscale toxicity of COOH-f-MWCNTs for proteins, which is a critical issue for in vivo application of COOH-f-MWCNTs., Competing Interests: The authors have declared that no competing interests exist.

Candida antarctica lipase B (CALB) belongs to psychrophilic lipases which hydrolyze carboxyl ester bonds at low temperatures. There have been some features reported about cold-activity of the enzyme through experimental methods, whereas there is no detailed information on its mechanism of action at molecular level. Herein, a comparative molecular dynamics simulation and essential dynamics analysis have been carried out at three temperatures (5, 35 and 50°C) to trace the dominant factors in the psychrophilic properties of CALB under cold condition. The results clearly describe the effect of temperature on CALB with meaningful differences in the flexibility of the lid region (a5 helix), covering residues 141-147. Open- closed conformations have been obtained from different sets of long-term simulations (60 ns) at 5°C gave two reproducible distinct forms of CALB. The starting open conformation became closed immediately at 35 and 50°C during 60 ns of simulation, while a sequential open-closed form was observed at 5°C. These structural alterations were resulted from α5 helical movements, where the closed conformation of active site cleft was formed by displacement of both helix and its side chains. Analysis of normal mode showed concerted motions that are involved in the movement of both α5 and α10 helices. It is suggested that the functional motions needed for lypolytic activity of CALB is constructed from short-range movement of α5, accompanied by long-range movement of the domains connected to the lid region. [ABSTRACT FROM AUTHOR]