High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase

Abstract

Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressuredenaturation process of IPMDH.

High-pressure protein crystallography of hen egg-white lysozyme

“…Collins et al (2005) reported water filling ISSN 1399-0047 of the nonpolar interior cavity of L99A mutant T4 lysozyme. As the initial steps of pressure denaturation, Nagae et al (2012) also observed water penetration into the hydrophobic cavity at the dimer interface and a cleft on the surface of IPMDH. To further study high-pressure effects in proteins, including pressure-induced changes in hydration structure, we used HPPX to study the well known tetragonal crystal of HEWL.…”

“…To collect high-completeness data sets at each pressure, we inserted two or three crystals into the DAC at different orientations. To prevent the crystals from moving in the DAC sample chamber, the crystals were fixed with tied fibres from cigarette filters (Nagae et al, 2012). The pressure in the DAC was gradually increased to the target pressure in about 10-15 min, and the DAC was left for about 10 min.…”

“…For example, the use of DACs permitted structural studies to be made of bovine copper zinc superoxide dismutase (SOD) at 1 GPa (Fourme et al, 2001) and of Cowpea mosaic virus at 330 MPa (Girard et al, 2005). Our group also developed a HPPX environment at the Photon Factory, Japan (Chavas et al, 2013), with which we studied the pressure-induced structural changes of 3-isopropylmalate dehydrogenase (IPMDH) up to 650 MPa (Nagae et al, 2012). In particular, the results for SOD and IPMDH show that the pressure-induced structural changes observed by HPPX are directly related to protein activity or function.…”

A molecular perspective on the limits of life: Enzymes under pressure

“…As in pressure induced protein unfolding, water may play a role in oligomer dissociation by pressure due to a "destruction of voids" mechanism. For instance, crystallographic studies at different pressures of the dimeric (∼ 340 residues/monomer) enzyme 3-isopropylmalate dehydrogenase (IPMDH), a pressure sensitive enzyme in the biosynthesis pathway of leucine, from the mesophilic Shewanella oneidensis [50] shows water inside a cavity at the interface of the two monomers between 4.1 to 5.8 kbar while no water is present at 1 bar ( figure 3). This cavity may be a pressure sensitive point for dimer dissociation.…”

“…The results from these simulations also indicate that µs simulations are needed to evaluate the changes in structural properties at different pressures since very low frequency motions are apparent. High-pressure crystallographic studies of monomeric and dimeric proteins have been used to estimate the compressibility to be between 4 to 6 Mbar −1 [50,53,54], and internal cavities within the monomers of IPMDH have been shown to be compressed monotonously up to 6.5 kbar [50], indicating that the cavities allow the protein to be more compressible.…”