5 research outputs found
Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor
Phospholipases
A2 (PLA2) catalyze the hydrolysis reaction of sn-2
fatty acids of membrane phospholipids and are also involved in receptor
signaling and transcriptional pathways. Here, we used pressure modulation
of the PLA2 activity and of the membrane’s physical–chemical
properties to reveal new mechanistic information about the membrane
association and subsequent enzymatic reaction of PLA2. Although the
effect of high hydrostatic pressure (HHP) on aqueous soluble and integral
membrane proteins has been investigated to some extent, its effect
on enzymatic reactions operating at the water/lipid interface has
not been explored, yet. This study focuses on the effect of HHP on
the structure, membrane binding and enzymatic activity of membrane-associated
bee venom PLA2, covering a pressure range up to 2 kbar. To this end,
high-pressure Fourier-transform infrared and high-pressure stopped-flow
fluorescence spectroscopies were applied. The results show that PLA2
binding to model biomembranes is not significantly affected by pressure
and occurs in at least two kinetically distinct steps. Followed by
fast initial membrane association, structural reorganization of α-helical
segments of PLA2 takes place at the lipid water interface. FRET-based
activity measurements reveal that pressure has a marked inhibitory
effect on the lipid hydrolysis rate, which decreases by 75% upon compression
up to 2 kbar. Lipid hydrolysis under extreme environmental conditions,
such as those encountered in the deep sea where pressures up to the
kbar-level are encountered, is hence markedly affected by HHP, rendering
PLA2, next to being a primary osmosensor, a good candidate for a sensitive
pressure sensor in vivo
Molecular Determinants of Expansivity of Native Globular Proteins: A Pressure Perturbation Calorimetry Study
There
is a growing interest in understanding how hydrostatic pressure
(<i>P</i>) impacts the thermodynamic stability (Δ<i>G</i>) of globular proteins. The pressure dependence of stability
is defined by the change in volume upon denaturation, Δ<i>V</i> = (∂Δ<i>G</i>/∂<i>P</i>)<sub><i>T</i></sub>. The temperature dependence of change
in volume upon denaturation itself is defined by the changes in thermal
expansivity (Δ<i>E</i>), Δ<i>E</i> = (∂Δ<i>V</i>/∂<i>T</i>)<sub><i>P</i></sub>. The pressure perturbation calorimetry (PPC)
allows direct experimental measurement of the thermal expansion coefficient,
α = <i>E</i>/<i>V</i>, of a protein in the
native, α<sub>N</sub>(<i>T</i>), and unfolded, α<sub>U</sub>(<i>T</i>), states as a function of temperature.
We have shown previously that α<sub>U</sub>(<i>T</i>) is a nonlinear function of temperature but can be predicted well
from the amino acid sequence using <i>α</i>(<i>T</i>) values for individual amino acids (<i>J. Phys. Chem.
B </i><b>2010</b>, <i>114</i>, 16166–16170).
In this work, we report PPC results on a diverse set of nine proteins
and discuss molecular factors that can potentially influence the thermal
expansion coefficient, α<sub>N</sub>(<i>T</i>), and
the thermal expansivity, <i>E</i><sub>N</sub>(<i>T</i>), of proteins in the native state. Direct experimental measurements
by PPC show that α<sub>N</sub>(<i>T</i>) and <i>E</i><sub>N</sub>(<i>T</i>) functions vary significantly
for different proteins. Using comparative analysis and site-directed
mutagenesis, we have eliminated the role of various structural or
thermodynamic properties of these proteins such as the number of amino
acid residues, secondary structure content, packing density, electrostriction,
dynamics, or thermostability. We have also shown that α<sub>N</sub>(<i>T</i>) and <i>E</i><sub>N,sp</sub>(<i>T</i>) functions for a given protein are rather insensitive
to the small changes in the amino acid sequence, suggesting that α<sub>N</sub>(<i>T</i>) and <i>E</i><sub>N</sub>(<i>T</i>) functions might be defined by a topology of a given protein
fold. This conclusion is supported by the similarity of α<sub>N</sub>(<i>T</i>) and <i>E</i><sub>N</sub>(<i>T</i>) functions for six resurrected ancestral thioredoxins
that vary in sequence but have very similar tertiary structure
Design principles for high–pressure force fields: Aqueous TMAO solutions from ambient to kilobar pressures
Accurate force fields are one of the major pillars on which successful molecular dynamics simulations of complex biomolecular processes rest. They have been optimized for ambient conditions, whereas high-pressure simulations become increasingly important in pressure perturbation studies, using pressure as an independent thermodynamic variable. Here, we explore the design of non-polarizable force fields tailored to work well in the realm of kilobar pressures - while avoiding complete reparameterization. Our key is to first compute the pressure-induced electronic and structural response of a solute by combining an integral equation approach to include pressure effects on solvent structure with a quantum-chemical treatment of the solute within the embedded cluster reference interaction site model (EC-RISM) framework. Next, the solute's response to compression is taken into account by introducing pressure-dependence into selected parameters of a well-established force field. In our proof-of-principle study, the full machinery is applied to N,N,N-trimethylamine-N-oxide (TMAO) in water being a potent osmolyte that counteracts pressure denaturation. EC-RISM theory is shown to describe well the charge redistribution upon compression of TMAO(aq) to 10 kbar, which is then embodied in force field molecular dynamics by pressure-dependent partial charges. The performance of the high pressure force field is assessed by comparing to experimental and ab initio molecular dynamics data. Beyond its broad usefulness for designing non-polarizable force fields for extreme thermodynamic conditions, a good description of the pressure-response of solutions is highly recommended when constructing and validating polarizable force fields. (C) 2016 AIP Publishing LLC