Pressure Denaturation of Staphylococcal Nuclease Studied by Neutron Small-Angle Scattering and Molecular Simulation

Abstract

We studied the pressure-induced folding/unfolding transition of staphylococcal nuclease (SN) over a pressure range of approximately 1-3 kilobars at 25 degrees C by small-angle neutron scattering and molecular dynamics simulations. We find that applying pressure leads to a twofold increase in the radius of gyration derived from the small-angle neutron scattering spectra, and P(r), the pair distance distribution function, broadens and shows a transition from a unimodal to a bimodal distribution as the protein unfolds. The results indicate that the globular structure of SN is retained across the folding/unfolding transition although this structure is less compact and elongated relative to the native structure. Pressure-induced unfolding is initiated in the molecular dynamics simulations by inserting water molecules into the protein interior and applying pressure. The P(r) calculated from these simulations likewise broadens and shows a similar unimodal-to-bimodal transition with increasing pressure. The simulations also reveal that the bimodal P(r) for the pressure-unfolded state arises as the protein expands and forms two subdomains that effectively diffuse apart during initial stages of unfolding. Hydrophobic contact maps derived from the simulations show that water insertions into the protein interior and the application of pressure together destabilize hydrophobic contacts between these two subdomains. The findings support a mechanism for the pressure-induced unfolding of SN in which water penetration into the hydrophobic core plays a central role.

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

“…On the basis of experimental studies and simulations (Frye & Royer, 1998;Paliwal et al, 2004), these effects are currently considered to be mainly caused by the penetration of water molecules into the protein interior under high pressure. The solution structure of ubiquitin determined at 300 MPa by high-pressure NMR spectroscopy (Kitahara et al, 2005) was marked by a conformational change from a closed to an open form of ubiquitin.…”

On the Denaturation Mechanisms of the Ligand Binding Domain of Thyroid Hormone Receptors

“…Proteins such as barnase, 32 BPTI, 33 the engrailed homeodomain, 27 and the villin headpiece subdomain, 34 among others, 28,35,36 for which the denaturation mechanisms have been studied by computer simulations, do not exhibit complete elements of the secondary structure protected from solvent. Moreover, knowledge of the denaturation pathways of the LBDs may help to identify residues that are important for large-amplitude motions of the protein and, therefore, contributes to better understanding the molecular basis of the diseases caused by their mutations.…”

Pressure-induced Structural and Hydration Changes of Proteins in Aqueous Solutions

“…The water in the hydration shell of proteins has been proposed to play a crucial role in high pressure induced protein unfolding, and it was proposed that the penetration of water molecules into the hydrophobic core may induce unfolding of the system. 26 The water penetration model was also supported by high pressure molecular dynamics simulations. 27 Winter et al 28 carried out a series of molecular dynamics computer simulations on SNase.…”