Abstract
Here, we report results obtained during our experiments to visualize how heat transforms globular protein, lysozyme into building block of β-amyloids. Light scattering experiments showed formation of lower order associated species around 50–70 °C followed by rapid cooperativity to β-amyloid fibrils. Interestingly, crystallization drops set at higher temperatures either led to aggregates or spherulites. The latter possess an amorphous β-fibril rich core with thin crystalline needles projecting outwards. Diffraction of the crystalline outgrowths revealed novel dimers and trimers of lysozyme where individual chains were similar to monomer with marginal gain in β-sheet content. Importantly, analysis of Amide I stretching frequencies showed that protein loses its secondary structure at temperatures higher than where we obtained crystals followed by rapid gain in β-sheet content. Interestingly, attempts to use the needles as seeds for more crystals led to “broom-like” fibril formations at the ends. Further, aggregation inhibitors like arginine and benzyl alcohol completely obliterated spherulites formation during crystallization. Refinement of crystals of lysozyme in presence of these molecules showed these small molecules bind to the interfaces of heat associated dimers and trimers. Overall our work concludes that heat induced weakly associated structures of lysozyme are the first step towards its amyloid formation.
Highlights
We report results obtained during our experiments to visualize how heat transforms globular protein, lysozyme into building block of β-amyloids
Lara et al showed that lysozyme undergoes hydrolysis at pH 2 and elevated temperatures close to 90 °C, and implied that the products of hydrolysis induce the amyloidic fibril formation[6]
Spherulitic structures were observed in case of bovine insulin under low pH and high temperature (65 °C) conditions as well as HEWL in the presence of sodium nitrate and CSIR-Institute of Microbial Technology, Chandigarh, India
Summary
The increase in temperature reduces affinity of these ligands to lysozyme and/or increased diffusion dynamics dislodges these molecules It is very likely a complex summation of events in solution, but it remains clear from our repetitive attempts of crystallization of lysozyme at increased temperatures in presence of these molecules that no spherulites are formed indirectly supporting that the dimeric and trimeric states of lysozyme are precursors and “on pathway” ensembles of temperature induced amyloid formation by lysozyme. Another important aspect was the stability of the lysozyme protein under increased temperatures during crystallization attempts. Initial stock solutions were prepared by mixing the protein in 50 mM NaOAc buffer pH 3.8 containing 150 mM NaCl at concentrations ~30 mg/ml and dialyzed overnight
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
