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; F. LUIS GONZÁLEZ FLECHA
Circular Dichroism: Theory and Spectroscopy
Lugar: New York; Año: 2011;
This chapter discusses the use of far and near UV circular dichroism methods to analyze changes in secondary andtertiary structure during protein unfolding and how to obtain thermodynamic and kinetic information. We will give abrief introduction on the basics of the technique and discuss practical examples on the analysis of the unfolding ofpeptides, 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 thedetermination of the pre and post transition baselines. The importance of steady-state and time-resolved CDmeasurements 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 quantitativedescription of the unfolding transition. Whereas folding-unfolding kinetics (non-equilibrium experiments) will giveus 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 andmulti-state protein folding transitions, pointing to the determination and proper interpretation of thermodynamic andkinetic 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, andthioredoxin, and the thermophilic membrane protein CopA from Archaeoglobus fulgidus. Also, induction of helicalstructure by co-solvents (e. g. TFE, SDS) and their stability will be discussed.