Lysozyme Interaction with Phospholipid Nanodroplets Probed by Sum Frequency Scattering Vibrational Spectroscopy

Abstract

When a nanoparticle (NP) is introduced into a biological environment, its identity and interactions are immediately attributed to the dense layer of proteins that quickly covers the particle. The formation of this layer, dubbed the protein corona, is in general a combination of proteins interacting with the surface of the NP and a contest between other proteins for binding sites either at the surface of the NP or upon the dense layer. Despite the importance for surface engineering and drug development, the molecular mechanisms and structure behind interfacial biomolecule action have largely remained elusive. We use ultrafast sum frequency scattering (SFS) spectroscopy to determine the structure and the mode of action by which these biomolecules interact with and manipulate interfaces. The majority of work in the field of sum frequency generation has been done on flat model interfaces. This limits some important membrane properties such as membrane fluidity and dimensionalityimportant factors in biomolecule–membrane interactions. To move toward three-dimensional (3D) nanoscopic interfaces, we utilize SFS spectroscopy to interrogate the surface of 3D lipid monolayers, which can be used as a model lipid-based nanocarrier system. In this study, we have utilized SFS spectroscopy to follow the action of lysozyme. SFS spectra in the amide I region suggest that there is lysozyme at the interface and that the lysozyme induces an increased lipid monolayer order. The binding of lysozyme with the NP is demonstrated by an increase in acyl chain order determined by the ratio of the CH3 symmetric and CH2 symmetric peak amplitudes. Furthermore, the lipid headgroup orientation s-PO2 – change strongly supports lysozyme insertion into the lipid layer causing lipid disruption and reorientation. Altogether, with SFS, we have made a huge stride toward understanding the binding and structure change of proteins within the protein corona.

Oil-in-Water Emulsions Probed Using Fluorescence Multivariate-Curve-Resolution Spectroscopy

“…60−62 2% hexadecane is added to an aqueous solution that was mixed for 5 min with a hand-held homogenizer (TH, OMNI International) and subsequently placed in an ultrasonic bath (35 kHz, 400 W, Bandelin) for the same duration. 38,63,64 The average size, size distribution (PDI), and zeta-potential of the nanodroplets were measured with dynamic light scattering (DLS, Malvern ZS nanosizer). The nanodroplets were consistently found to have a mean diameter in the range of 200−300 nm with a polydispersity index (PDI) of less than 0.25.…”

“…Oil nanoemulsions which consist of bare oil nanodroplets were prepared in aqueous solution with a known protocol of was prepared with anionic surfactants sodium dodecyl sulfonate (SDS) and sodium dodecylbenzenesulfonate (SDBS) and with cationic surfactants cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide hydrate (CBH) at different concentrations below their critical micelle concentration. − 2% hexadecane is added to an aqueous solution that was mixed for 5 min with a hand-held homogenizer (TH, OMNI International) and subsequently placed in an ultrasonic bath (35 kHz, 400 W, Bandelin) for the same duration. ,, The average size, size distribution (PDI), and zeta-potential of the nanodroplets were measured with dynamic light scattering (DLS, Malvern ZS nanosizer). The nanodroplets were consistently found to have a mean diameter in the range of 200–300 nm with a polydispersity index (PDI) of less than 0.25.…”

Disulfide-Driven Charge and Hydrophobicity Rearrangement of Remodeled Membrane Proteins toward Amyloid-Type Aggregation

“…As a powerful analytical technique suited for biointerface detection under nonvacuum conditions, sum frequency generation (SFG) vibrational spectroscopy has been widely employed to study interfacial proteins and lipid membranes, offering structural and dynamic information at the molecular level in situ in real time. − The SFG process is intrinsically surface/interface-selective, as it is typically permitted at surfaces/interfaces where inversion symmetry is broken, rendering SFG highly sensitive to the biointerfaces associated with structural asymmetry. ,− Lysozyme, as a positively charged precursor protein with four disulfide bonds, was selected as a model protein for their potential to undergo disulfide-based restructuring and amyloid-type aggregation at interfaces. ,, Tris(2-carboxy­ethyl)phosphine (TCEP) used in this system can reduce disulfide bonds to dithiols, thus inducing the transformation of lysozyme from an oxidized folding state to a reduced folding state . As demonstrated in the previous SFG studies, lysozyme aggregated at the lipid monolayer interface in the low-pH environment , or only structured at the air/water, solid/water, or nanodroplet interfaces. − However, lysozyme aggregation by the reductive pathway on lipid bilayer membranes, which is relatively efficient and physiological, , has not been fully appreciated and remains to be clarified, due to a lack of molecular-level understanding of the interfacial structures and the aggregation kinetics, along with the influence on transmembrane asymmetry. Herein, we applied SFG to investigate the disulfide-reduction-induced lysozyme remodeling process on lipid membranes in a physiological aqueous solution (Figure S1), instead of the aberrantly nonphysiological conditions (such as extreme pH and heating).…”