High-Pressure FTIR Studies of the Secondary Structure of Proteins

Abstract

Infrared spectra of five globular proteins (bovine pancreas ribonuclease A, horse skeletal muscle myoglobin, bovine pancreas insulin, horse heart cytochrome c, egg white lysozyme) in 5% D2O solutions (pD 7.0) were measured as a function of pressure up to 1470 MPa at 30 °C. According to the second-derivative spectral changes in the observed amide I band of the proteins, which indicate that the α-helix and β-sheet substructures of the secondary structures break dramatically into the random coil conformation, ribonuclease A and myoglobin are denatured reversibly at 850 MPa and 350 MPa, respectively. Lysozyme denatures partially and reversibly at 670 MPa, as shown by decrease in the α-helix and β-turn substructures, but no change occurs in the random coil and β-sheet substructures. The secondary structure of cytochrome c is not disrupted at pressures up to 1470 MPa, and partial transformation of the α-helix of insulin to random coil starts at 960 MPa. Hydrogen-deuterium exchange of protons on the amide groups in the protein interior is increased by external pressure and is associated with the pressure-induced protein conformational changes. A number of studies on the effects of pressure on protein denaturation have been carried out using various high-pressure detection methods: ultraviolet absorbance spectroscopy (Brandts et al., 1970; Hawley, 1971), visible absorbance spectroscopy (Zipp & Kauzmann, 1973), fluorescence intensity spectroscopy (Li et al., 1976), polarization fluorescence spectroscopy (Chryssomallis et al., 1981), and enzyme activity assays (Taniguchi & Suzuki, 1983; Makimoto et al., 1989). These techniques have the great advantage of being applicable to pressure-induced reversible denaturation of proteins to identify the thermodynamic parameters, especially the volume change and compressibility of a protein in solution, because the experiments can be run under dilute conditions at a protein concentration of less than 0.05% w/v. Therefore, these data reflect the intramolecular phenomena of reversible pressure changes and provide the volume changes accompanying the denaturation of proteins, which are due to the difference in partial molal (specific) volume between the native and denatured proteins in solution.