Süleyman Cinar

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 membranes 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.

Secondary structure and folding stability of proteins adsorbed on silica particles – Pressure versus temperature denaturation

We present a systematic study of the pressure and temperature dependent unfolding behavior of proteins that are adsorbed on silica particles. Hen egg white lysozyme and bovine ribonuclease A (RNase) were used as model proteins, and their secondary structures were resolved by Fourier transform infrared (FTIR) spectroscopy in the temperature range of 10-90°C and the pressure range of 1-16,000bar. Apparently, the secondary structures of both proteins do not change significantly when they are adsorbing on the silica particles. Remarkably, the changes of the secondary structure elements upon protein unfolding are very similar in the adsorbed and the free states. This similarity could be observed for both lysozyme and RNase using both high pressures and high temperatures as denaturing conditions. However, the pressures and temperatures of unfolding of lysozyme and RNase are drastically lowered upon adsorption indicating lower folding stabilities of the proteins on the silica particles. Moreover, the temperature ranges, where changes in secondary structure occur, are broadened due to adsorption, which is related to smaller enthalpy changes of unfolding. For both proteins, free or adsorbed, pressure-induced unfolding always leads to less pronounced changes in secondary structure than temperature-induced unfolding. In the case of lysozyme, high pressure also favors a different unfolded conformation than high temperature. Overall, the results of this study reveal that adsorption of proteins on silica particles decreases the folding stability against high pressures and temperatures, whereas the unfolding pathways are mainly preserved in the adsorbed state.

Effect of interfacial properties on the activation volume of adsorbed enzymes.

We have studied the enzymatic activities of α-chymotrypsin (α-CT) and horseradish peroxidase (HRP) that are adsorbed on various chemically modified planar surfaces under aqueous solution. The enzymes were adsorbed on bare quartz, hydrophobic poly(styrene) (PS), positively charged poly(allylamine hydrochloride) (PAH), and negatively charged poly(styrene sulfonate) (PSS). Activation volumes of the enzymes at the aqueous-solid interfaces were determined by using high-pressure total internal reflection fluorescence (TIRF) spectroscopy. Apparently, the pressure response of the adsorbed enzymes strongly depends on the interfacial properties. α-CT can be activated by pressure (increasing enzymatic rate) on negatively charged surfaces like quartz and PSS, whereas HRP is activated by pressure on hydrophobic PS. Corresponding negative activation volumes of -29 mL mol(-1) for α-CT on quartz, -23 mL mol(-1) for α-CT on PSS, and -35 mL mol(-1) for HRP on PS are found. In addition, the absolute activities of α-CT and HRP on quartz, PS, PAH and PSS were determined by UV absorption at ambient pressure. Remarkably, large activities are found on those surfaces that are associated with negative activation volumes. However, Fourier transform infrared (FTIR) spectra collected in attenuated total reflection (ATR) mode do not indicate major adsorption induced conformational changes of the enzymes at any interface studied. Overall, the results of this study show that the activity of immobilized enzymes can largely be enhanced by the right combination of adsorbent material and applied pressure.

Building polyelectrolyte multilayers with calmodulin – a neutron and X-ray reflectivity study

We have studied the formation and functional properties of polyelectrolyte multilayers where calmodulin (CaM) is used as a polyanion. CaM is known to populate distinct conformational states upon binding Ca2+ and small ligand molecules. Therefore, we have also probed the effects of Ca2+ ions and trifluoperazine (TFP) as ligand molecule on the interfacial structures. Multilayers with the maximum sequence PEI-(PSS-PAH)x-CaM-PAH-CaM-PAH have been deposited on silicon wafers and characterized by X-ray and neutron reflectometry. From the analysis of all data, several remarkable conclusions can be drawn. When CaM is deposited for the second time, a much thicker sublayer is produced than in the first CaM deposition step. However, upon rinsing with PAH, very thin CaM-PAH sublayers remain. There are no indications that ligand TFP can be involved in the multilayer buildup due to strong CaM-PAH interactions. However, there is a significant increase in the multilayer thickness upon removal of Ca2+ ions from holo-CaM and an equivalent decrease in the multilayer thickness upon subsequent saturation of apo-CaM with Ca2+ ions. Presumably, CaM can still be toggled between an apo and a holo state, when it is embedded in polyelectrolyte multilayers, providing an approach to design bioresponsive interfaces.

Pressure‐Induced Dissolution and Reentrant Formation of Condensed, Liquid–Liquid Phase‐Separated Elastomeric α‐Elastin

We investigated the combined effects of temperature and pressure on liquid-liquid phase separation (LLPS) phenomena of α-elastin up to the multi-kbar regime. FT-IR spectroscopy, CD, UV/Vis absorption, phase-contrast light and fluorescence microscopy techniques were employed to reveal structural changes and mesoscopic phase states of the system. A novel pressure-induced reentrant LLPS was observed in the intermediate temperature range. A molecular-level picture, in particular on the role of hydrophobic interactions, hydration, and void volume in controlling LLPS phenomena is presented. The potential role of the LLPS phenomena in the development of early cellular compartmentalization is discussed, which might have started in the deep sea, where pressures up to the kbar level are encountered.

Inhibitor and peptide binding to calmodulin characterized by high pressure Fourier transform infrared spectroscopy and Förster resonance energy transfer

We compare the binding of an inhibitor with that of a natural peptide to Ca2+ saturated calmodulin (holo-CaM). As inhibitor we have chosen trifluoperazine (TFP) that is inducing a huge conformational change of holo-CaM from the open dumbbell-shaped to the closed globular conformation upon binding. On the other hand, melittin is used as model peptide, which is a well-known natural binding partner of holo-CaM. The experiments are carried out as a function of pressure to reveal the contribution of volume or packing effects to the stability of the calmodulin-ligand complexes. From high-pressure Fourier transform infrared (FTIR) spectroscopy, we find that the holo-CaM/TFP complex has a much higher pressure stability than the holo-CaM/melittin complex. Although the analysis of the secondary structure of holo-CaM (without and with ligand) indicates no major changes up to several kbar, pressure-induced exposure of α-helices to water is most pronounced for holo-CaM without ligand, followed by holo-CaM/melittin and then holo-CaM/TFP. Moreover, structural pressure resistance of the holo-CaM/TFP complex in comparison with the holo-CaM/melittin complex is also clearly visible by higher Ca2+ affinity. Förster resonance energy transfer (FRET) from the Tyr residues of holo-CaM to the Trp residue of melittin even suggests some partial dissociation of the complex under pressure which points to void volumes at the protein-ligand interface and to electrostatic binding. Thus, all results of this study show that the inhibitor TFP binds to holo-CaM with higher packing density than the peptide melittin enabling a favorable volume contribution to the inhibitor efficiency.

Bioresponsive interfaces composed of calmodulin and poly(ethylene glycol): Toggling the interfacial film thickness by protein-ligand binding

Responsive interfaces are often realized by polymer films that change their structure and properties upon changing the pH-value, ionic strength or temperature. Here, we present a bioresponsive interfacial structure that is based on a protein, calmodulin (CaM), which undergoes a huge conformational change upon ligand binding. At first, we characterize the conformational functionality of a double Cys mutant of CaM by small-angle X-ray scattering (SAXS) and Fourier transform infrared (FTIR) spectroscopy. The CaM mutant is then used to cross-link poly(ethylene glycol) (PEG) chains, which are bound covalently to a supporting planar Si surface. These films are characterized by X-ray reflectometry (XR) in a humidity chamber providing full hydration. It is well known that Ca2+-saturated holo-CaM binds trifluoperazine (TFP) and changes its conformation from an open, dumbbell-shaped to a closed, globular one in solution. At the interface, we observe an increase of the PEG-CaM film thickness, when TFP is binding and inducing the closed conformation, whereas the removal of Ca2+-ions and a concomitant release of TFP is associated with a decrease of the film thickness. This toggling of the film thickness is largely reversible. In this way, a structural change of the interface is achieved via protein functionality which has the advantage of being selective for ligand molecules without changing the environmental conditions in a harsh way via physico-chemical parameters.

Secondary Structure and Folding Stability of Proteins Adsorbed on Silica - Pressure Versus Temperature Denaturation

In an ongoing project, we are studying the combined effects of interfaces and high pressure on proteins. So far, we have obtained some insight into the volume changes of protein adsorption, the pressure stability, and the activation volumes of adsorbed enzymes. Apparently, protein adsorption is enhanced under high pressure conditions due to a pressure-induced destabilization of the protein native state. Moreover, the secondary structures of lysozyme and RNase do not change significantly when they are adsorbing on the silica particles. Remarkably, the changes of the secondary structure elements upon protein unfolding are very similar in the adsorbed and the free states. This similarity could be observed for both lysozyme and RNase using both high pressures and high temperatures as denaturing conditions. However, the pressures and temperatures of unfolding of lysozyme and RNase are drastically lowered upon adsorption indicating lower folding stabilities of the proteins on the silica particles. For both proteins, free or adsorbed, pressure-induced unfolding always leads to less pronounced changes in secondary structure than temperature-induced unfolding. Overall, the results of this study reveal that adsorption of proteins on silica particles decreases the folding stability against high pressures and temperatures, whereas the unfolding pathways are mainly preserved in the adsorbed state.