Pressure-induced molten globule state of human acetylcholinesterase: structural and dynamical changes monitored by neutron scattering.

We used small-angle neutron scattering (SANS) to study the effects of high hydrostatic pressure on the structure of human acetylcholinesterase (hAChE). At atmospheric pressure, our SANS results obtained on D11 at ILL (Grenoble, France) give a radius of gyration close to that calculated for a mixture of monomers, dimers and tetramers of the enzyme, suggesting a good agreement between hAChE crystal structure and its conformation in solution. Applying high pressure to the sample we found a global compression of about 11% of the enzyme up to a pressure of 900 bar and then again an extension up to 2.1 kbar indicating unfolding of the tertiary structure due to a molten globule (MG) state. On the other hand, we studied the influence of pressure up to 6 kbar on the dynamics of this enzyme, on the backscattering spectrometer IN13 at ILL. For the first time, we used elastic incoherent neutron scattering (EINS) to probe the differences between hAChE in its folded state (N), its high-pressure induced MG state and its unfolded state (U). Especially around the MG state at 1750 bar we found a significant increase in the dynamics, indicating a partial unfolding. A four-step-model is suggested to describe the changes in the protein.

Correlation of the dynamics of native human acetylcholinesterase and its inhibited huperzine A counterpart from sub-picoseconds to nanoseconds.

It is a long debated question whether catalytic activities of enzymes, which lie on the millisecond timescale, are possibly already reflected in variations in atomic thermal fluctuations on the pico- to nanosecond timescale. To shed light on this puzzle, the enzyme human acetylcholinesterase in its wild-type form and complexed with the inhibitor huperzine A were investigated by various neutron scattering techniques and molecular dynamics simulations. Previous results on elastic neutron scattering at various timescales and simulations suggest that dynamical processes are not affected on average by the presence of the ligand within the considered time ranges between 10 ps and 1 ns. In the work presented here, the focus was laid on quasi-elastic (QENS) and inelastic neutron scattering (INS). These techniques give access to different kinds of individual diffusive motions and to the density of states of collective motions at the sub-picoseconds timescale. Hence, they permit going beyond the first approach of looking at mean square displacements. For both samples, the autocorrelation function was well described by a stretched-exponential function indicating a linkage between the timescales of fast and slow functional relaxation dynamics. The findings of the QENS and INS investigation are discussed in relation to the results of our earlier elastic incoherent neutron scattering and molecular dynamics simulations.

Effects of hydrostatic pressure on horse liver alcohol dehydrogenase (HLADH): a new way of analyzing kinetic study.

Oxidation of ethanol by horse liver alcohol dehydrogenase (HLADH) is monitored under pressure (0.1 MPa - 225 MPa). The pressure-induced modifications of catalytic activity are followed by plotting reaction velocities as a function of substrates concentrations in the traditional double reciprocal form: then, pressure is treated as an activator (p < 100 MPa) or an inhibitor (p<225 MPa). Surprising typical patterns of Lineweaver-Burk curves are observed and interpreted. These results suggest that this approach could be a powerful tool to study enzyme's structure-activity relationship.