Protein Conformational Intermediates Characterized with Novel High Pressure EPR and CD Techniques

Abstract

Regulation of protein function is often linked to conformational intermediates that exist in equilibrium with the ground state. In many cases, these intermediate states exist as only a small fraction of the total protein conformational ensemble. It has been shown that high hydrostatic pressure is an invaluable tool for populating low-lying, excited states to levels amenable to spectroscopic detection. Here we demonstrate the usefulness of high pressure for populating such states as monitored by two novel techniques: High pressure electron paramagnetic resonance spectroscopy (EPR) and high pressure circular dichroism (CD). High pressure EPR was used in conjunction with site-directed spin labeling to monitor changes in local protein structure with applied pressure (up to 400 MPa). The relative partial molar volume and isothermal compressibility of each conformational substate was determined from the pressure dependence of the equilibrium constant determined from the continuous wave EPR spectra. High pressure CD was employed to detect global changes in secondary and tertiary structure at elevated pressure (up to 200 MPa). The combination of site-specific and global information provided by these techniques provides a more complete description of pressure excited intermediate states. Data from apomyoglobin and a cavity containing mutant (L99A) of T4 lysozyme are presented.