The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two-state transition.
Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolution-alternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations.
Xu
Ming
M
Department of Chemistry, University at Albany, SUNY, Albany, New York 12222, USA.
Shashilov
Victor A
VA
Ermolenkov
Vladimir V
VV
Fredriksen
Laura
L
Zagorevski
Dmitri
D
Lednev
Igor K
IK
D000818:Animals; D002645:Chickens; D005260:Female; D007700:Kinetics; D015233:Models, Statistical; D009113:Muramidase / Q000737:chemistry*; D044367:Phase Transition; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; C421864:hen egg lysozyme; D009113:Muramidase