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>)_<i>T</i>. 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>)_<i>P</i>. 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_N(<i>T</i>), and unfolded, α_U(<i>T</i>), states as a function of temperature. We have shown previously that α_U(<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_N(<i>T</i>), and the thermal expansivity, <i>E</i>_N(<i>T</i>), of proteins in the native state. Direct experimental measurements by PPC show that α_N(<i>T</i>) and <i>E</i>_N(<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_N(<i>T</i>) and <i>E</i>_N,sp(<i>T</i>) functions for a given protein are rather insensitive to the small changes in the amino acid sequence, suggesting that α_N(<i>T</i>) and <i>E</i>_N(<i>T</i>) functions might be defined by a topology of a given protein fold. This conclusion is supported by the similarity of α_N(<i>T</i>) and <i>E</i>_N(<i>T</i>) functions for six resurrected ancestral thioredoxins that vary in sequence but have very similar tertiary structure