Detection of temperature-induced molten globule states in small, β-sheet-rich proteins by infrared spectroscopy.

The study of the mechanisms underlying formation of molten globule states in proteins is gaining growing interest due to recent discoveries concerning the involvement of this folding intermediate in several biological processes and to the possibility it offers to clarify the general process of protein folding. Different spectroscopic techniques, such as fluorescence, circular dichroism, nuclear magnetic resonance and infrared, have proved to be invaluable tools in the detection and characterization of molten globule states. In this review, the use of Fourier Transform infrared (FT-IR) spectroscopy to shed light on the formation of molten globules is discussed. Due to its ability to simultaneously probe the secondary structure and compactness/flexibility of protein samples prepared in deuterated media, FT-IR spectroscopy is particularly well suited to detect temperature-induced molten globule states in small, β-sheet-rich proteins. Several approaches for analysis and interpretation of infrared spectra are provided, such as second derivatives, Amide I′ position and extent of H/D exchange plots, difference spectra and 2D correlation analysis. Experimental cases are also provided as examples.