Pressure Modulation of the Enzymatic Activity of. Phospholipase A2, a Putative Membraneassociated

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1 SUPPORTING INFORMATION Pressure Modulation of the Enzymatic Activity of Phospholipase A2, a Putative Membraneassociated Pressure Sensor Saba Suladze, Suleyman Cinar, Benjamin Sperlich, and Roland Winter* Department of Chemistry and Chemical Biology, Biophysical Chemistry, TU Dortmund University, Dortmund, Otto-Hahn-Str. 6, D Dortmund, Germany * To whom correspondence should be addressed. roland.winter@tu-dortmund.de. Atomic Force Microscopy (AFM) - experimental details. The preparation of the supported lipid bilayers and the AFM setup is described in detail in refs. S1,S2. For the enzyme membrane interaction studies, 25 L of PLA2 (c PLA2 =.35 µm) in Tris buffer (2 mm Tris, 7 mm MgCl 2, 2 mm CaCl 2 or.5 mm EGTA, ph 7.4) were injected into the AFM fluid cell and allowed to incubate for 1 h at room temperature. Measurements were performed at room temperature using a MultiMode scanning probe microscope with a NanoScope IIIa controller (Digital Instruments (now Bruker), Santa Barbara, USA) and a J-Scanner (scan size 125 m). Images were obtained by applying the tapping mode in liquid with sharp nitride lever (SNL) probes mounted in a fluid cell (MTFML, both Veeco (now Bruker), Mannheim, Germany). Tips with nominal force S1

2 constants of.24 N m 1 were used at driving frequencies around 9 khz and drive amplitudes between 2 and 4 mv; scan frequencies were between 1. and 2. Hz. The images with resolutions of pixels were analyzed using the image analysis and processing software NanoScope version 5 and 6 (Veeco (now Bruker), Mannheim, Germany) and Origin 8.6 (OriginLab, Northampton, USA). Lipid vesicles were prepared from stock solutions of 1 mg ml 1 lipid (DOPC, DOPG, DPPC, DPPG, and Chol) in chloroform/methanol 4:1 for DPPG and in chloroform for all other lipids and mixed to obtain 1.94 mg of total lipid with the composition of DOPC/DOPG/DPPC/DPPG/Chol 2:5:45:5:25 (mol%). After removal of the solvent by drying under vaccum overnight, the dry lipids were resuspended in 1 ml of 2 mm Tris, 7 mm MgCl 2, 2 mm CaCl 2, ph 7.4 (hydrolysis expected) and in 1 ml of 2 mm Tris, 7 mm MgCl 2,.5 mm EGTA, ph 7.4 (negative control experiment: no hydrolysis expected) for the AFM experiments to yield a total lipid concentration of 3 mm. Details on the formation of large unilamellar vesicles (LUVs) of 1 nm size by extrusion are described in S3. All buffers were filtrated and sonicated. AFM imaging of bvpla2 activity. Heterogeneous model membrane systems such as DOPC/DOPG/DPPC/DPPG/Chol have been widely used in studies to gain detailed information about membrane protein interactions. S1,S2,S4 The five component membrane anionic raft-like system leads to segregation into liquid-ordered and liquid-disordered phases and mimics the heterogeneity of biological membranes. To reveal the behavior of PLA2 upon membrane interaction on a nanometer length scale with an imaging technique, time-lapse tapping mode atomic force microscopy (AFM) experiments were carried out (Figure S4). The interaction between the enzyme and membrane was followed by imaging the same membrane region at different time points. Lipid spreading forms coexistence of liquid-ordered (l o phase) and liquiddisordered (l d phase) phases with a height difference of approximately 1 nm between l o and l d domains, which is in good agreement with previous studies (Figure S4a), S1,S3 showing that the thickness of the lipid bilayer is approximately 5.2 nm for the l o -phase and 4. nm for the l d - phase. S3 Time-lapse tapping-mode AFM experiments were then carried out first after injection of the enzyme solution under noncatalytic conditions (25 µl of.35 µm PLA2 in the presence.5 mm EGTA in buffer solution) to observe possible rearrangements of lipid phases upon PLA2 S2

3 insertion into the lipid bilayer. As can be seen from Figure S4b, no significant changes of the membrane s lateral organization are visible upon bvpla2 binding. Membrane hydrolysis of the catalytically active enzyme was then monitored in the presence of 2 mm CaCl 2, revealing hydrolysis of a major part of the membrane already within 1 min after injection (Figure S4c). Moreover, the typical height difference of 1 nm between the l d - and l o -phase is no more detectable, implying destruction of the lipid phase co-existence. After 24 hours, the effect of the PLA2 activity on the membrane structure is even more dramatic. Height differences of about 3.7 nm indicate appearance of a remaining thinner disordered phase which is not homogeneously spreading the mica surface anymore, i.e., hole formation is invoked as well. Simonsen et al. pointed out that in case of phase coexistence, PLA2 preferentially hydrolyses the l d -phase, but a high amount of cholesterol protects lipid bilayers against complete destruction. S4 S3

4 Additional Figures a) b) Fourier self-deconvolution spectra 1676 cm cm -1 to 1656 cm cm cm -1 1 bar 5.2 kbar 9.7 kbar 1614 cm Fourier self-deconvolution spectra 1656 / 1649 cm cm -1 1 bar 1676 cm cm -1 4 kbar 1678 / 167 cm -1 1 kbar to 1614 cm cm wavenumber / cm -1 wavenumber / cm -1 c) absorbance wavenumber / cm -1 1 bar 1 bar Figure S1. Conformational changes in bvpla2 upon pressurization at 37 C (a) in bulk buffer solution and (b) in the presence of the lipid bilayer in the pressure range from 1 bar to 1 kbar. (c) Comparison of amide-i' spectra of bvpla2 in bulk solution at 1 bar before and after pressurization, indicating full recovery of the native protein structure. S4

5 Normalized fluoroscence anionic raft k 1 = 38.9 s -1 A 1 =.613 au k 2 = 3.29 s -1 A 1 =.379 au DOPC/DOPG k 1 = 37.7 s -1 A 1 =.888 au k 2 = 7.25 s -1 A 2 =.13 au t / s Figure S2. Representative association curve of.35 μm bvpla2 to liposomes of anionic raftlike and DOPC/DOPG bilayers at a 62.5 μm lipid concentration. Normalized quantum yield p / bar Figure S3. Pressure dependence of the fluorescence intensity of the β-dph-hpc fluorophore. Pressure induced changes on the fluorescence of pure β-dph-hpc labelled anionic raft-like lipid vesicles (c lipids =.125 mm, c β-dph-hpc = 5 μm, ex: 34 nm, em: 435nm) at 37 C. The steadystate fluorescence intensity data are normalized to the 1 bar fluorescent intensity value. S5

6 a) b) c) d) l d l d l o l o nm nm nm nm -3.5 nm µm -4. nm µm -4. nm µm -4. nm µm Figure S4. AFM visualization of lipid bilayers with coexisting l o and l d domains of a heterogeneous DOPC/DOPG/DPPC/DPPG/Chol (2:5:45:5:25 mol%) lipid bilayer membrane on mica at 25 o C of (a) the membrane only, (b) after 1 min of bvpla2 injection in the presence of EGTA, (c) after 1 min of hydrolysis, and (d) after 24 h of hydrolysis. Corresponding section profile of the AFM images are given below each image. Arrows display height differences between l o /l d phases (a-b) or thicknesses of holes in bilayers as a consequence of the hydrolysis reaction (c-d). S6

7 Table S1. Secondary structure content of BvPLA2 at ambient pressure as determined by X-ray diffraction in comparison with the FTIR spectroscopy data obtained in this study (accuracy: 2%). Please note that owing to differences in transition dipole moments of the different secondary structure elements, full agreement of the X-ray and FTIR data cannot be expected. Rather, relative pressure-induced changes will essentially be discussed. Secondary structure element BvPLA2 from X-ray diffraction (1POC) BvPLA2 in bulk solution from FTIR BvPLA2 in the presence of membrane from FTIR α-helices 29.1 % 35.4 % 39.8 % β-sheets 22.4 % 26.6 % 27.7 % turns 31.3 % 19.4 % 15.4 % unordered/random 17.2 % 18.6 % 17.1 % References (S1) Weise, K.; Kapoor, S.; Denter, C.; Nikolaus, J.; Opitz, N.; Koch, S.; Triola, G.; Herrmann, A.; Waldmann, H.; Winter, R. J. Am. Chem. Soc. 211, 133, (S2) Weise, K.; Triola, G.; Brunsveld, L.; Waldmann, H.; Winter, R. J. Am. Chem. Soc. 29, 131, (S3) Kapoor, S.; Werkmuller, A.; Denter, C.; Zhai, Y.; Markgraf, J.; Weise, K.; Opitz, N.; Winter, R. Biochim. Biophys. Acta 211, 188, (S4) Simonsen, A. C. Biophys. J. 28, 94, S7