Kazumi Hata

Pressure-induced chemical shifts as probes for conformational fluctuations in proteins.

2013 Elsevier B.V. All rights reserved.

Structural Plasticity of Staphylococcal Nuclease Probed by Perturbation with Pressure and pH

The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. Small angle X‐ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys‐66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys‐66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys‐66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (<2 kbar) at pH values where Lys‐66 is normally neutral at ambient pressure left most of the structure unperturbed but produced significant nonlinear changes in chemical shifts in the helix where Lys‐66 is located. Above 2 kbar pressure at these pH values the protein with Lys‐66 unfolded cooperatively adopting a relatively compact, albeit random structure according to Kratky analysis of the SAXS data. In contrast, at low pH and high pressure the unfolded state of the variant with Lys‐66 is more expanded than that of the reference protein. The combined global and local view of the structural reorganization triggered by ionization of the internal Lys‐66 reveals more detectable changes than were previously suggested by NMR spectroscopy at ambient pressure. Proteins 2011.

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80°C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53ml/mol at 25°C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.