Infrared spectroscopy and ultrasonic velocimetry as a tool for probing conformational flexibility of proteins

Abstract

Because of the crucial importance of structural fluctuations for function and stability of proteins, there is a strong interest for the relationships between structural fluctuations, the parameters of protein denaturation and the kinetics of H/D-exchange. Structural fluctuations can be described by volume and entropy fluctuations and these quantities are accessible via the isothermal compressibility, the thermal expansion and the isobaric heat capacity.

Our aim is to present the principal problem concerning the experimental procedures to answer those questions using lysozyme and α-lactalbumin. Whereas the transition parameters and the kinetics of the H/D-exchange were obtained using FTIR spectroscopy, the adiabatic compressibility was obtained by a combination of ultrasonic velocimetry and densitometry. It could be shown that the stability of the investigated proteins is correlated with reduced volume fluctuations. The expected direct correlation between H/D exchange rates and structural fluctuations could not be seen and it is assumed that the interactions are more complex than from the intuitive point of view.

Key Words:

• Protein compressibility
• structural fluctuations
• denaturation
• H/D exchange
• acoustic interferometry
• densitometry