INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
capítulos de libros
The use of circular dichroism methods to monitor unfolding transitions in peptides, globular and membrane proteins
ERNESTO A. ROMAN; JAVIER SANTOS; LUIS GONZÁLEZ FLECHA
Circular Dichroism: Theory, Spectroscopy, and Advances in Nan
Lugar: Hauppauge, NY, EEUU.; Año: 2010;
This chapter discusses the use of far and near UV circular dichroism methods to analyze changes in secondary and tertiary structure during protein unfolding and how to obtain thermodynamic and kinetic information. We will give a brief introduction on the basics of the technique and discuss practical examples on the analysis of the unfolding of peptides, globular and membrane proteins. Here we will deal with important issues such as protein concentration, path length selection, choice of buffer, wavelength selection, and specific issues for unfolding experiments as the determination of the pre and post transition baselines. The importance of steady-state and time-resolved CD measurements on protein folding studies will be pointed out. Near- and far-UV CD experiments under equilibrium-condition will aid us in the characterization of folded, partially folded, and unfolded states and in the quantitative description of the unfolding transition. Whereas folding-unfolding kinetics (non-equilibrium experiments) will give us a clue about the dynamics of the involved process. We will give a walkthrough to perform experiments and quantitatively analyze them in terms of the two-state and multi-state protein folding transitions, pointing to the determination and proper interpretation of thermodynamic and kinetic parameters. In addition, the advantages of using this method and its limitations will be discussed. The selected examples will be focused on the unfolding of the soluble proteins β-lactamase, lysozyme, and thioredoxin, and the thermophilic membrane protein CopA from Archaeoglobus fulgidus. Also, induction of helical structure by co-solvents (e. g. TFE, SDS) and their stability will be discussed.