Adsorption of Denaturated Lysozyme at the Air–Water Interface: Structure and Morphology

Abstract

The application of protein deuteration and high flux neutron reflectometry has allowed a comparison of the adsorption properties of lysozyme at the air-water interface from dilute solutions in the absence and presence of high concentrations of two strong denaturants: urea and guanidine hydrochloride (GuHCl). The surface excess and adsorption layer thickness were resolved and complemented by images of the mesoscopic lateral morphology from Brewster angle microscopy. It was revealed that the thickness of the adsorption layer in the absence of added denaturants is less than the short axial length of the lysozyme molecule, which indicates deformation of the globules at the interface. Two-dimensional elongated aggregates in the surface layer merge over time to form an extensive network at the approach to steady state. Addition of denaturants in the bulk results in an acceleration of adsorption and an increase of the adsorption layer thickness. These results are attributed to incomplete collapse of the globules in the bulk from the effects of the denaturants as a result of interactions between remote amino acid residues. Both effects may be connected to an increase of the effective total volume of macromolecules due to the changes of their tertiary structure, that is, the formation of molten globules under the influence of urea and the partial unfolding of globules under the influence of GuHCl. In the former case, the increase of globule hydrophobicity leads to cooperative aggregation in the surface layer during adsorption. Unlike in the case of solutions without denaturants, the surface aggregates are short and wormlike, their size does not change with time, and they do not merge to form an extensive network at the approach to steady state. To the best of our knowledge, these are the first observations of cooperative aggregation in lysozyme adsorption layers.

DNA Interaction with a Polyelectrolyte Monolayer at Solution—Air Interface

“…The strong distinctions in the induction period are even observed for solutions with identical concentrations of the components ( Figure S2a,b, Supporting Information ). The insufficient reproducibility of the induction period was already observed for some systems and can be connected with the poorly controlled processes of the nucleation and the subsequent growth of surface aggregates [ 48 ]. The surface properties start to change only when the size and/or surface concentration of the aggregates increase and they start to interact.…”

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

“…The expression of D-HEWL PP was achieved as described by Campbell et al (2018). Since the protein was secreted into the extracellular medium, the supernatant was recovered upon cell pelleting.…”

“…The recombinant production of HEWL in Escherichia coli is challenged by the reductive environment of the bacterial cytosol, which prevents the correct formation of its four disulfide bridges, resulting in the formation of inclusion bodies. The use of the yeast Pichia pastoris has been investigated as an expression system for the production of recombinant HEWL (Liu et al, 2003;Li et al, 2012;Mine et al, 1999;Campbell et al, 2018). While this approach results in the production of high-quality protein, the low yield is problematic for neutron crystallographic and spectroscopic applications.…”

Recent advances in resolving kinetic and dynamic processes at the air/water interface using specular neutron reflectometry

“…In combination with ellipsometry and Brewster angle microscopy measurements, they showed that the maximum size of a Bragg diffraction peak from specular NR is related to a growth-collapse mechanism: first the film gets thicker driven by an evaporative steady state, and then CO 2 adsorption causes its breakup. Also, Noskov and co-workers recently exploited protein deuteration to resolve information about the adsorption kinetics, structure and morphology of lysozyme monolayers at the air/water interface in the absence and presence of high bulk concentrations of strong denaturants [63]. They showed that the denaturants result in an acceleration of adsorption and an increase of the adsorption layer thickness, which together were attributed to incomplete collapse of the globules in the bulk.…”