Structure Determination of Hen Egg-White Lysozyme Aggregates Adsorbed to Lipid/Water and Air/Water Interfaces.

S Strazdaite,E Navakauskas,G Niaura,J Kirschner,T Sneideris

doi:10.1021/acs.langmuir.9b03826

Abstract

We use vibrational sum-frequency generation (VSFG) spectroscopy to study the structure of hen egg-white lysozyme (HEWL) aggregates adsorbed to DOPG/D2O and air/D2O interfaces. We find that aggregates with a parallel and antiparallel β-sheet structure together with smaller unordered aggregates and a denaturated protein are adsorbed to both interfaces. We demonstrate that to retrieve this information, fitting of the VSFG spectra is essential. The number of bands contributing to the VSFG spectrum might be misinterpreted, due to interference between peaks with opposite orientation and a nonresonant background. Our study identified hydrophobicity as the main driving force for adsorption to the air/D2O interface. Adsorption to the DOPG/D2O interface is also influenced by hydrophobic interaction; however, electrostatic interaction between the charged protein's groups and the lipid's headgroups has the most significant effect on the adsorption. We find that the intensity of the VSFG spectrum at the DOPG/D2O interface is strongly enhanced by varying the pH of the solution. We show that this change is not due to a change of lysozyme's and its aggregates' charge but due to dipole reorientation at the DOPG/D2O interface. This finding suggests that extra care must be taken when interpreting the VSFG spectrum of proteins adsorbed at the lipid/water interface.

Highlights

A vast amount of proteins can self-assemble into highly ordered, predominantly β-sheet-rich structures that are generally named as amyloids.[1−3] In vivo, amyloids are associated with a range of neurodegenerative diseases, of which Alzheimer’s and Parkinson’s diseases are the most known.[4]
It was shown that amyloid-β (Aβ) that is linked to Alzheimer’s disease preferentially adsorbs to lipid/water interfaces and starts to form fibrillar aggregates.[11−13] Analogously, the same was observed for α-synuclein,[14] insulin,[15,16] human islet amyloid polypeptide,[17,18] lysozyme,[19,20] and other proteins
Aliquots from the lysozyme solution were taken out and, first, investigated by Atomic Force Microscopy (AFM) in order to check the morphology of aggregates and to confirm the formation of lysozyme fibrils and by FTIR, to determine the structure of the aggregates formed in the bulk solution

Summary

Introduction

A vast amount of proteins can self-assemble into highly ordered, predominantly β-sheet-rich structures that are generally named as amyloids.[1−3] In vivo, amyloids are associated with a range of neurodegenerative diseases, of which Alzheimer’s and Parkinson’s diseases are the most known.[4]. Recent studies have shown that small oligomers and fragmented fibrils have more potential to induce membrane damage.[22] For example, smaller oligomeric aggregates of Aβ were shown to exhibit a cytotoxic effect, while large fibrillar aggregates did not trigger cell injury.[23] it was reported that smaller Aβ aggregates have preferential adsorption over the larger ones.[24] Small onpathway α-synuclein aggregates can insert into the negatively charged lipid bilayer and form larger coaggregates from protein and lipid, damaging the membrane irreversibly.[25]

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Conclusion