UV-CD12: synchrotron radiation circular dichroism beamline at ANKA

UV-CD12 at ANKA and its current end-station are described, with a standard module for vacuum-UV synchrotron radiation circular dichroism of bio-macromolecules in the liquid state, and a unique module for macroscopically oriented lipid membranes (oriented circular dichroism).

with the same measurement parameters and the same data post-processing as for the (KIGAKI)3 experiments (see below). For converting the spectral data into molar circular dichroism units (∆ε), the exact concentration of the protein solution was determined based on the UV absorption at 280 nm. A molar extinction coefficient of 13980 L mol -1 cm -1 was used for myoglobin (Pace et al., 1995), which contains two Trp and two Tyr residues in its amino acid sequence.
SRCD measurements of human serum albumin (HSA) were performed to proof the absence of UVinduced denaturation (see Fig. 3C). HSA (SIGMA-Aldrich, Schnelldorf, Germany) was dissolved in water at a concentration of 3.33 mg ml -1 and measured in a CaF2 demountable cell with 12.1 µm optical path length. Twenty consecutive single scans were recorded in direct succession over a wavelength range from 260-175 nm at maximum beam current of 160 mA and using the same measurement parameters and data post-processing as for the myoglobin and (KIGAKI)3 measurements (cf. section S1.7). In another series of measurements a second HSA sample of the same concentration was measured with the same measurement parameters, but in this case the sample was exposed to the SRCD beam at 170 nm for 10 min. before each consecutive scan (see Fig. S6), which are harsh conditions never applied in a typical user experiment.
For preparation of the final CD samples, aliquots of the peptide stock solutions in water were added to the liposome dispersion. The final peptide concentration in the (KIGAKI)3 vesicle dispersions was around 135 µM, and the lipid concentration was 6.75 mM, resulting in a peptide-to-lipid (P/L) ratio of 1:50. S1.7. Thermal ramping SRCD experiments of (KIGAKI)3 UV-CD12 was used to measure SRCD spectra of the (KIGAKI)3 peptide and a mutant in which Ile-8 was replaced by the non-natural fluorinated D-amino acid CF3-Bpg (3-[trifluoromethyl]-D-bicyclopent-[1.1.1]-1-ylglycine), in a DMPC/DMPG 3:1 lipid dispersion in water. A 0.1 mm fixed path length cylindrical quartz glass cell (Hellma, Müllheim, Germany) was used for the thermal scan SRCD experiments, with the aim of minimizing the background absorption of these samples resulting from H2O and scattering artifacts caused by the lipid vesicles, which lead to increased noise levels in CD spectra measured at a bench-top instrument especially at wavelengths < 200 nm. Demountable cells with an even thinner optical path could not be used for these experiments, because the sample would dry out at higher temperatures due to the evaporation of water. With the help of the cylindrical cell it was possible to measure both peptides in lipid vesicles reliably down to 180 nm even at elevated temperatures. For the SRCD thermal scans the samples were thermostated at temperatures ranging from 30-80°C in 10° steps, and back using a 1 K min -1 heating/cooling rate, and a 5 min delay time at each temperature equilibration step between successive measurements. The temperature of the SR-LCD module described in section 2.2 of the paper and the thermal ramping process were automatically controlled by using an in-house data-acquisition software developed under the LabView environment.
Under the SRCD conditions it was sufficient to collect only two scans from 260-180 nm at 0.5 nm intervals, using a fixed 1 nm spectral bandwidth, a 0.3 s lock-in time, a 1.5 s dwell time, and a scan-rate of 17 nm min -1 . SRCD spectra were processed with CDTool software, i.e. the averaged baseline of a peptide-free lipid reference sample measured at 30°C was subtracted from the averaged sample spectra of the thermal run, and finally the data were smoothened by a Savitzky-Golay algorithm contained in this software package (Lees at al., 2004).

S1.8. Cloning, expression and purification of PDGFRβ-TM domain, sample preparation of oriented lipid bilayers and SR-OCD measurements
The 39 amino acid stretch of the PDGFRβ transmembrane domain was produced recombinantly at the IBG-2. Cloning, expression and purification have been described in detail previously (Muhle-Goll et al., 2012). For OCD, the PDGFRβ transmembrane segment had to be reconstituted in oriented lipid bilayers.
First, the lyophilized protein and 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC, Avanti Polar Lipids, Alabaster, AL, USA) lipid powder were co-solubilized in chloroform/methanol 1:1, and insoluble parts were removed by centrifugation. An aliquot of the protein/lipid solution was deposited onto a quartz glass plate, and the solution was allowed to dry in air. The amount of peptide and lipid on the plate was 1.58 and 79 nanomoles, respectively, with a P/L ratio of 1:50. For the second sample with a P/L of 1:500 the amount of peptide and lipid on the plate was 0.6 and 300 nanomoles. The samples then were placed under vacuum to remove residual organic solvents, followed by subsequent rehydration at 97% relative humidity overnight in the OCD cell shown in Fig. 2B, to obtain macroscopically aligned oriented lipid bilayers. OCD measurements on the J-810 desktop spectropolarimeter were performed as described earlier (Bürck et al., 2008, Muhle-Goll et al., 2012 in the wavelength range from 260-180 nm, using a 20 nm min -1 scan rate, an automated slit with nominal 1 nm spectral bandwidth, and a 4 s response time. At each of the 8 rotation angles one scan was acquired, and afterwards the average was taken, and the rotationally averaged reference spectrum of the pure lipid was subtracted to obtain the final spectrum.
SR-OCD measurements of the same sample and in the same OCD cell at UV-CD12 were performed in the spectral range from 260-180 nm, using a scan-rate of 20 nm min -1 and comparable acquisition parameters, i.e. a lock-in time of 0.3 s, and a dwell time of 1.5 s, using a fixed spectral band width of 1 nm. For all OCD measurements the temperature of the sample was set to 30°C in order to stay above the lipid phase transition temperature.
For the control experiments on the J-810 instrument at different exit slit widths (see Fig. S7) a PDGFRβ/DEiPC sample with a P/L ratio of 1:50 was measured at each setting of the slit width (0.8, 1.4, 1.8, 2.2, 2.6, and 3.0 mm) using the same conditions as described above. However, no lipid baseline reference was measured and subtracted as these measurements were just intended to show the reduced signal magnitude of the OCD spectrum at increasing slit width.
For another control experiment with the J-810 due to lack of PDGFRβ peptide OCD spectra of the helical antimicrobial peptide PGLa in DErPC lipid bilayers (1,2-dierucanoyl-sn-glycero-3phosphocholine, Avanti Polar Lipids, Alabaster, AL, USA) were measured at a P/L of 1:100. Here, a black cardboard with a punched out pinhole of 2.5 x 4.8 mm which mimicked the UV-CD12 beam geometry was centered in front of the OCD cell and spectra with and without pinhole were measured using the same measurement parameters as above. The corresponding OCD spectra are shown in Fig.   S8.

S1.9. Dried myoglobin film sample
The measurement with the dried myoglobin film was performed using a flat CaF2 plate as a support, which absorbs only below 130 nm. To measure the dried spectrum, the CaF2 plate was fitted into the OCD cell holder at ambient humidity to maintain a stable orientation in the incident light beam. The plate was carefully cleaned with milliQ water and dried before a baseline spectrum was collected.
Subsequently, 6 µL of a 1.265 mg mL -1 horse myoglobin solution in H2O (0.4 nMol) were deposited on the CaF2 plate and gently dried in air. Afterwards, the sample was dried under low vacuum (about 15 mbar) for 5 min and visually inspected for particles or opaque areas. The sample was transferred to the SR-OCD setup, and a sample spectrum was obtained in the wavelength range from 280-170 nm using the standard acquisition parameters shown in Table 1. A second series of measurement was done from 175-130 nm, using a lock-in time of 1 s, a dwell time of 5 s, and a scan rate of 5 nm min -1 . For obtaining a good signal-to-noise ratio at this low wavelength range, a total of 16 scans (2 at each of the 8 rotation angles) were averaged. Finally, the averaged spectrum of the empty CaF2 plate was subtracted, and the two spectra from 280-170 and 175-130 nm were concatenated to give the final spectrum.  Wavelength calibration below 160 nm with the absorption spectrum of dry nitrogen used for purging the sample chamber, data in brackets are reference values from (Trickle et al.,1995).

Figure S3
A 0.02% solution of toluene in hexane was measured at different exit slit widths. The peak ratio of the maximum to the minimum absorbance is a measure of the spectral band width, which is given in the table (inset).

Figure S4
Transmission spectra of standard salt solutions were recorded on UV-CD12 to measure the level of polychromatic stray light, which is typically in a range between 0.1 and 0.2%.

Figure S8
Comparison of OCD spectra of the α-helical antimicrobial peptide PGLa in fully hydrated oriented DErPC lipid bilayers (P/L 1:100) measured on the J-810 with and without pinhole in front of the OCD cell (the pinhole mimicks the UV-CD12 beam size). Here no lipid reference was subtracted. The signal magnitude is enhanced by ~40% when the pinhole is inserted, which demonstrates that the 4.3-fold bigger beam size of the J-810 hits also inhomogeneous "empty" zones at the outer rim of the sample which leads on average to a signal reduction compared with the UV-CD12 beam size geometry. For the J-810 measurement an increased noise level is obtained at wavelengths < 200 nm with the pinhole inserted because less photons reach the detector due to the reduced beam size.