The dynamic nature of incubation solution after cooling to room temperature in amyloid formation of hen egg white lysozyme: An FTIR assessment

Abstract

Amyloid formation of hen egg white lysozyme (HEWL) usually requires elevated temperature, while biophysical characterizations on the incubation solution are often performed at room temperature. Whether maintaining the incubation solution at room temperature results in further structural changes is a significantly important issue that has never been explored. Herein, we use FTIR spectroscopy to assess this issue and reveal that the hot incubation solution of HEWL after cooling to room temperature is in a dynamically evolving state and forms β-sheet aggregates continuously over time. Combined with AFM, we show that these aggregates are non-fibrillar β-sheet aggregates and have vibrational signature distinct from that of fibrillar aggregates. Using FTIR difference spectroscopy, we demonstrate that these non-fibrillar aggregates are in an anti-parallel β-sheet configuration. We also provide a detailed discussion on the spectral assignments for protein aggregates in anti-parallel and parallel β-sheet configurations. With FTIR second derivative technique, we show that these non-fibrillar aggregates are in fact present along with fibrillar aggregates during incubation under elevated temperature but are less stable compared with that at room temperature. Implications from the current work are discussed.