Probing the structural basis and adsorption mechanism of an enzyme on nano-sized protein carriers

Abstract

Silica nanoparticles (SiNPs) are important nano-sized, solid-state carriers/hosts to load, store, and deliver biological or pharmaceutical cargoes. They are also good potential solid supports to immobilize proteins for fundamental protein structure and dynamics studies. However, precaution is necessary when using SiNPs in these areas because adsorption might alter the activity of the cargoes, especially when enzymes are loaded. Therefore, it becomes important to understand the structural basis of the cargo enzyme activity changes, if there is any. The high complexity and dynamics of the nano-bio interface present many challenges. Reported here is a comprehensive study of the structure, dynamics, and activity of a model enzyme, T4 lysozyme, upon adsorption to a few surface-modified SiNPs using several experimental techniques. Not surprisingly, a significant activity loss on each studied SiNP was found. The structural basis of the activity loss was identified based on results from a unique technique, the Electron Paramagnetic Resonance (EPR) spectroscopy, which probes structural information regardless of the complexity. Several docking models of the enzyme on SiNPs with different surfaces, at different enzyme-to-SiNP ratios are proposed. Interestingly, we found that the adsorbed enzyme can be desorbed via pH adjustment, which highlighted the potential to use SiNPs for enzyme/protein delivery or storage due to the high capacity. In order to use SiNPs as enzyme hosts, minimizing the enzymatic activity loss upon adsorption is needed. Lastly, the work outlined here demonstrate the use of EPR in probing structural information on the complex (inorganic)nano-bio interface.

Study of Protein Dynamics under Nanoconfinement by Spin-Label ESR: A Case of T4 Lysozyme Protein

“…Previously T4L, when adsorbed at solid surface such as quartz 44 or silica nanoparticles 45 , was found to become partially unfolded and lose its enzymatic activity, but the present study indicates that T4L retains its conformation when adsorbed inside nanochannels. Therefore, it is important to perform a careful inspection of the protein properties, when proteins are loaded onto surface of nanoparticles or into nanochannels, using the demonstration methods of the present study.…”

Spin‐labeling of polymeric micelles and application in probing micelle swelling using EPR spectroscopy

“…Upon confirming the formation of micelles, CW EPR was used to investigate the regional dynamics of the micelles at the labeled site (the core‐shell interface). EPR line shape is known to be sensitive to dynamics, or more precisely, the rotational diffusion and the associated spatial ordering, of the spin label in a manner that the faster the label's rotational diffusion rate is and/or the less spatial limitation is, the narrower the EPR linewidth. In contrast, the slower the rate is and/or the more spatially restricted the motion is, the broader the spectrum.…”

Insights on the Structure, Molecular Weight and Activity of an Antibacterial Protein–Polymer Hybrid

“…The mutants of T4L were expressed, purified, and spin labeled using the previously reported procedure (for details see the Supplementary Information, SI). [48] The commercial hydroxylated PEGs were purchased from Sigma Aldrich. The details of polymer modification and polymer-protein conjugation are provided in the SI.…”

“…One promising approach to remove these technical barriers is Electron Paramagnetic Resonance (EPR) spectroscopy.E PR has been widely used in probing structure and conformational dynamics in complex, large biological or polymeric systems. [36][37][38][39][40][41][42][43][44][45][46] Recently,wehave reported EPR in probing structural insights at the complex nano-bioi nterfaces [47,48] and in polymeric micelles. [49] EPR can probe structural information in the native stateo ft he target system,r egardless of thes ize and complexity.I na ddition, once spin labeled, the hybrid's concentrationi sp roportional to the spin concentrationw hich is immune of any complications caused by polymer optical ab-Protein-polymer conjugates are attractive biomaterials which combine the functions of both proteins and polymers.…”