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

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.

Membrane-Protein-Hydration Interaction of α-Synuclein with Anionic Vesicles Probed via Angle-Resolved Second-Harmonic Scattering.

Amyloid formation of the protein α-synuclein promotes neurodegeneration in Parkinson's disease. The normal function of α-synuclein includes synaptic vesicle transport and fusion, and the protein binds strongly to negatively charged vesicles in vitro. Here, we demonstrate that nonresonant angle-resolved second-harmonic scattering detects α-synuclein binding to liposomes through changes in water orientational correlations and can thus be used as a high-accuracy and high-throughput label-free probe of protein-liposome interactions. The obtained results support a binding model in which the N-terminus of α-synuclein adopts an α-helical conformation that lies flat on the vesicle surface while the negatively charged C-terminus remains in solution.

Modified Biosensor for Cholinesterase Inhibitors with Guinea Green B as the Color Indicator.

Colorimetric biosensors of cholinesterase inhibitors are ideal for fast, reliable, and very simple detection of agents in air, in water, and on surfaces. This paper describes an innovation of the Czech Detehit biosensor, which is based on a biochemical enzymatic reaction visualized by using Ellman's reagent as a chromogenic indicator. The modification basically consists of a much more distinct color response of the biosensor, attained through optimization of the reaction system by using Guinea Green B as the indicator. The performance of the modified biosensor was verified on the chemical warfare agents (sarin, soman, cyclosarin, and VX) in water. The detection limits ascertained visually (with the naked eye) were about 0.001 µg/mL in water (exposure time 60 s, inhibition efficiency 25%).