Two-dimensional Raman and Raman optical activity correlation and factor analysis of lysozyme fibrillation

T. Pazderka¹, V. Kopecký Jr.¹, K. Hofbauerová^1,2, V. Baumruk¹

¹Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, Prague 2, CZ-121 16, Czech Republic

²Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4, CZ-142 20, Czech Republic

pazderka@karlov.mff.cuni.cz

Understanding of processes of amyloid fibrils formation is one of the key tasks in searching for proteins structural origin of human neurodegenerative diseases. Therefore, hen egg white lysozyme (HEWL) can serve as a good model of amyloid fibril formation. Furthermore, this protein is homologous to human lysozyme, which is one of the proteins that cause amyloid diseases [1]. Despite the intense scientific research, studying mechanisms in homologous proteins to lysozyme, the detailed mechanism of fibril formation is still far from complete understanding. Nevertheless, Raman optical activity (ROA) and Raman spectroscopy are very powerful techniques for studies of unfolded proteins. Promising experiments on lysozyme has already been done [2].

Here we present Raman and ROA study using 2D correlation spectroscopy (2DCoS) [3]. Firstly, we model changes of ROA band shapes and positions and investigate characteristic patterns in 2DCoS because the origin of 2D patterns for spectra with positive and negative bands has not been investigated yet. Subsequently, temporal and thermal (from 20 °C to 60 °C) spectral changes in ROA and Raman spectra of HEWL were analyzed by means of factor analysis and 2DCoS. It gave us an opportunity to study delicate details of HEWL fibrillation and denaturation, i.e. sequence of the secondary structure changes upon fibrils formation. Moreover, application of heterospectral 2DCoS enabled to transfer band assignment from Raman spectroscopy to ROA and spectral changes in conformation of S-S bridges were identified in protein ROA spectra for the first time.

References

1. D. R. Booth, M. Sunde, V. Bellotti et al., Nature, 385, (1997), 787.

2. E. W. Blanch, L. A. Morozova-Roche, D. A. E. Cochran et al., J. Mol. Biol., 301, (2000), 553.

3. I. Noda & Y. Ozaki, Two dimensional correlation spectroscopy: applications in vibrational and optical spectroscopy, (Chichester: Wiley), 2004.

Acknowledgements.

The Grant Agency of the Czech Republic is acknowledged for the support (projects 305/09/0457 and P205/10/1276).