High-Resolution 2D ¹H−¹⁵N NMR Characterization of Persistent Structural Alterations of Proteins Induced by Interactions with Silica Nanoparticles

Abstract

The binding of protein to solid surfaces often induces changes in the structure, and to investigate these matters we have selected two different protein-nanoparticle systems. The first system concerns the enzyme human carbonic anhydrase II which binds essentially irreversibly to the nanoparticles, and the second system concerns human carbonic anhydrase I which alternate between the adsorbed and free state upon interaction with nanoparticles. Application of the TROSY pulse sequence has allowed high-resolution NMR analysis for both of the protein-nanoparticle systems. For HCAII it was possible to observe spectra of protein when bound to the nanoparticles. The results indicated that HCAII undergoes large rearrangements, forming an ensemble of molten globule-like structures on the surface. The spectra from the HCAI-nanoparticle system are dominated by HCAI molecules in solution. A comparative analysis of variations in intensity from 97 amide resonances in a 1H-15N TROSY spectrum revealed the effects from interaction with nanoparticle on the protein structure at amino acid resolution.

Chemical Basis of Interactions Between Engineered Nanoparticles and Biological Systems

“…Unlike the “hard” (very stable) protein carbonic anhydrase II, the “soft” (less stable) protein carbonic anhydrase I undergoes a larger structural rearrangement when adsorbed onto silica nanoparticles, as shown by NMR. 171 The size of silica nanoparticles determines the surface curvature, which influences protein adsorption and the stability of the adsorbed proteins. For instance, larger nanoparticles cause a greater loss of α helicity and enzymatic activity in bound lysozyme 172 and induce a greater decrease in the thermodynamic stability of RNase A.…”

Analytical strategies for detecting nanoparticle–protein interactions

“…Besides these methods, nuclear magnetic resonance (NMR) should be able to provide data from all coupled H and N atoms in a protein 17, 59 . X-ray crystallography is also a preferred method to evaluate protein 3-D structure and protein-NP binding 58 .…”

Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles

“…The association rate constants of some complexes approach the diffusion-controlled limit, whereas conformational changes upon binding may slow down the process by orders of magnitude. Although most kinetic studies of adsorbed proteins concern extended surfaces of larger particles, reported time scales of exchange of adsorbed proteins from silica, polymer and TiO 2 nanoparticles range from 100 s to many hours (19)(20)(21)(22)(23)(24). The affinities and/or exchange rates depend on molecular details and the stability of the protein toward unfolding (19,25).…”