Supporting information 1 2 3 Volume 71 (2015) Supporting information for article: 4 5 6 7 8 Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials Arne Meyer, Karsten Dierks, Rana Hussein, Karl Brillet, Hevila Brognaro and Christian Betzel 9 10
Supporting information, sup-1 11 12 13 Table S1 Summary of micelles sizes of commonly used detergents measured with in situ DLS, applied in the context of this study. molecular weight CMC Average Number Detergent (g/mol) (mm) Peak (nm) Countrate (khz) 1 ANAPOE-C12E9 583.0 0.05 2 Octaethylene glycol monododecyl ether 538.8 0.09 3 ANAPOE-C12E8 539.0 0.09 4 ANAPOE-C13E8 553.0 0.1 5 n-dodecyl-β-d-maltopyranoside 510.6 0.15 6 n-dodecyl-α-d-maltopyranoside 510.6 0.17 7 CYMAL-7 522.5 0.19 8 ANAPOE-X-114 536.0 0.2 9 ANAPOE-C12E10 627.0 0.2 10 n-undecyl-β-d-thiomaltopyranoside 512.7 0.21 11 ANAPOE-X-100 647.0 0.23 12 Sucrose monododecanoate 524.6 0.3 13 CYMAL-6 508.5 0.56 14 n-undecyl-α-d-maltopyranoside 496.6 0.58 4.53 +/- 0.51 60.4 +/- 33 3.78 +/- 0.50 65.6 +/- 17.4 4.90 +/- 1.06 34.0 +/- 20.9 3.75 +/- 0.25 98.7 +/- 53.5 3.86 +/- 0.70 137.8 +/- 98.4 3.66 +/- 0.51 246.6 +/- 70.8 3.86 +/- 0.38 310.6 +/- 80.5 85.1 +/- 86.9 85.1 +/- 86.9 73.2 +/- 24.1 73.2 +/- 24.1 3.50 +/- 0.32 305.5 +/- 44.0 3.93 +/- 0.32 20.7 +/- 1.4 2.92 +/- 0.05 195.4 +/- 4.9 3.32 +/- 0.52 12.9 +/- 1.4 2.87 +/- 0.23 231.0 +/- 52.1
Supporting information, sup-2 15 n-undecyl-β-d-maltopyranoside 496.6 0.59 16 CYCLOFOS-7 363.3 0.62 17 Pentaethylene glycol monodecyl ether 406.6 0.07 18 n-decyl-α-d-maltopyranoside 482.6 1.8 19 ANAPOE-C10E6 423.0 0.9 3.28 +/- 0.24 500.8 +/- 35.0 3.82 +/- 0.86 175.1 +/- 108.9 10.34 +/- 1.72 178.6 +/- 193.6 2.41 +/- 0.28 178.0 +/- 5.2 8.22 +/- 1.63 197.4 +/- 620.5 20 n-dodecyl-n,n-dimethylamine-n-oxide (LDAO or DDAO) 510.6 1.0 1.93 +/- 0.47 55.8 +/- 1.8 21 ANAPOE-C10E9 555.0 1.3 22 n-decyl-β-d-maltopyranoside 482.6 1.8 23 CYMAL-5 494.5 2.4 24 n-nonyl-β-d-thiomaltopyranoside 484.6 3.2 25 Dimethyldecylphosphine oxide 218.3 4.66 26 n-nonyl-β-d-maltopyranoside 468.5 6.0 27 n-nonyl-β-d-glucopyranoside 306.4 6.5 28 CYMAL-4 480.5 7.5 29 Tetraatheylene glycol monooctyl ether 306.5 8.0 30 n-octyl-β-d-thiomaltopyranoside 470.6 8.5 31 Hexaethylene glcol monooctyl ether (C8E6) 394.5 10.0 2.94 +/- 0.09 111.5 +/- 2.7 2.91 +/- 0.09 674.8 +/- 20.0 3.12 +/- 0.21 840.0 +/- 77.3 2.98 +/- 0.11 179.9 +/- 18.6 3.10 +/- 0.35 74.2 +/- 7.3 3.02 +/- 0.22 1941.1 +/- 145.2 6.63 +/- 0.06 3433.0 +/- 71.6 2.93 +/- 0.08 735.5 +/- 17.3 4.48 +/- 0.50 1159.5 +/- 512.6 2.84 +/- 0.24 3101.7 +/- 299.5 2.70 +/- 0.37 472.0 +/- 62.2 32 ANAMEG-7 335.4 19.5 3.93 +/- 2879.6 +/- 365.4
Supporting information, sup-3 0.36 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Figure S1 Histograms of n-alkyl-β-d-maltopyranosides. The legend shows the R h of the most prominent peaks and the according calculated molecular weight (MW), within the number of measurements (N) and the relative integral of the scattered light intensity. The integral is normed to the largest integral (A = 1.000).
Supporting information, sup-4 39 40 41
Supporting information, sup-5 42 43
Supporting information, sup-6 44 45 46
Supporting information, sup-7 47 48 49 50 51 52 53 54 55 Figure S2 A: Step 1: Bacteriorhodopsin dissolved to a concentration of 20 mg/ml in 16 mm CHAPSO in 100 mm sodium chlorid and 20 mm sodium acetat, ph 5.0. B: Step 2: Bacteriorhodopsin at 20 mg/ml in 16 mm CHAPSO, 100 mm NaCl and 20 mm NaOAc, ph 5.0 and 2% Triton X-100 in 20 mm NaOAc. C: Step 3: BR at 20 mg/ml in 16 mm CHAPSO, 100 mm NaCl and 20 mm NaOAc, ph 5.0. 2% Triton X-100 in 20 mm NaOAc, after 30 min of centrifugation at 800 g. D: Step 4: BR at 2 mg/ml in 2% Triton X-100 in 20 mm sodium acetate, centrifuged 5 min 10.000 g. E: Step 5: BR at 2 mg/ml in 2% Triton X-100 in 20 mm sodium acetate, centrifuged for 35 min at 10.000 g. F: Step 6: BR at 2 mg/ml in 2% Triton X-100 in 20 mm sodium acetate, centrifuged 20 min at 16.000 g. 56 57
Supporting information, sup-8 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82
Supporting information, sup-9 83 Figure S3 SDS-PAGE von BR obtained after individual purifications steps. 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 SDS-Page Bacteriorhodopsin (BR) Lane 1 = Empty Lane 2 = Buffer CHAPSO and Triton X-100 supernatant from concentrator bottom volume Lane 3 = BR mit 16 mm CHAPSO und Triton X-100, PM pellet resuspended Lane 4 = Buffer, 2% Triton X-100 in 100 mm NaCl, 20 mm sodium acetate ph 5.0. supernatant from concentrator bottom volume Lane 5 = Buffer 16 mm CHAPSO, 2% Triton X-100, 100 mm NaCl, 20 mm sodium acetate, ph 5.0 supernatant from concentrator bottom volume Lane 6 = Step 6 supernatant from concentrator bottom volume Lane 7 = Step 6 BR 2 mg/ml in 2% Triton X-100, 20 mm sodium acetate, ph 5.0 Lane 8 = BR pellet as a control in Triton X-100, resuspended Lane 9 = Marker Lane 10 = Empty
Supporting information, sup-10 100 101 Figure S4 Reference DLS measurement of pure Triton X-100 in 20 mm sodium acetate at ph 5.0. 102 103
Supporting information, sup-11 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 Figure S5 M = Marker 1 = Control fusion protein 2 = Fusion protein containing TEV without Tridecyl-β-D-maltoside (TDM) 3 = Fusion protein containing TEV protease with Tridecyl-β-D- maltoside (application 1:1 drop) 4 = Fusion protein containing TEV protease with Tridecyl-β -D- maltoside (application 1:2 drop 9.8 mm) 5 = Fusion protein containing TEV protease with Tridecyl-β-D- maltoside (application 2:1 drop) 6 = Control of Tridecyl β-d- maltopyranoside SDS-PAGE. The DTT concentration of the Mbp-dHBx cleavage with TEV was performed at a ratio of 1:100 with overnight incubation in presence of TDM at 37 C. The concentration of the protein used was 2 mg/ml. M = Marker, Lane 1 = Control fusion protein, Lane 2 = Fusion protein with TEV, Lane 3 = Fusion protein with TEV and Tridecyl-β-D-maltoside (mixed 1:1), Lane 4 = Fusion protein with TEV and Tridecyl-β-D-maltoside (mixed 1:2)
Supporting information, sup-12 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 This SDS-PAGE shows in Lane 1, 2 and 3 bands of a 14 kda protein, which is interpreted as separated DHBx-protein. The proteolytic cleavage was incomplete indicated by the presence of a 56 kda band interpreted as remaining Mbp-DHBx-fusion protein. In presence of TDM at concentrations of 6.4 mm (Lane 3) and 9.8 mm (Lane 4) there is an obvious cleavage of the fusion protein indicated by 44 kda and 14 kda bands. Lane 3 (Figure 10) indicated that the sample corresponding to R h of 5.28 nm contains three proteins, one band correspond to a protein of ~60 kda which we interpreted as the MBPdHBx fusion protein (MBP-dHBx theoretical Mw is 56 kda) the other band is interpreted as the MBP cleavage product (42kDa) and the third one as the 14 kda dhbx cleavage product. Therefore we interpreted the 5.28 nm peak to be a PDC. The addition of TDM does not influence the TEV protase activity but the detergent supports the solubility of the cleaved protein. Also no band could be observed of pure TDM used as a control (Lane 6). The proteolytic cleaving efficiency is shown by a comparison of the lane 3 with insufficient TDM concentration (6.5 mm) to form the PDC and lane 4 with a sufficient TBM concentration (9.8 mm) to form the PDC. It indicates that the oligomer digestion is inefficient yielding a lower amount of the 14 kda dhbx-protein when the protein is oligomerized. The uncleaved fusion protein, with 60 kda, the 40 kda protein was interpreted as the remaining MBP after cleavage (MBP has a molecular weight of 42 kda) and a third protein of 14 kda corresponding to the expected product of dhbx. 140 141 142 143 144 145 146 147 148 149 150 151 152 153 The presence of the dhbx product could be further accomplished by subsequent sequence analysis by mass spectrometry (Supplementary Material, Figure 6). Tryptic peptides of the digested protein were spotted onto an Anchor Chip target (Bruker Daltonik GmbH, Bremen, Germany) with α-cyano-4-hydroxycinnamic acid as matrix and analyzed by MALDI- TOF/TOF mass analysis using an ultraflextreme mass spectrometer (Bruker Daltonik) equipped with a smartbeam-ii laser with a repetition rate of 2 khz. The acquisition software was FlexControl (version 3.3). Mass spectra were acquired in reflector mode and externally calibrated using a peptide calibration mixture II (Bruker Daltonik) with masses between m/z 1046 and m/z 3147. After baseline substraction and peak picking in the processing software (FlexAnalysis, version 3.3, Bruker Daltonik), sequencing has been performed in manually in FlexAnalysis with a mass accuracy of 0.5 Da. At some detergent concentration a denaturating effect on the protein might occur since as long aliphatc groups may weakening the protein internal hydrophobic interactions. However
111.965 210.960 63.848 284.922 428.948 386.986 584.019 820.179 673.122 743.127 1259.445 1372.536 1535.557 1622.575 1747.009 2018.939 511.980 1158.351 483.975 1030.310 2161.947 174.956 1693.725 Intens. [a.u.] Acta Cryst. (2015). F71, doi:10.1107/s2053230x14027149 Supporting information, sup-13 154 155 156 157 n-dodecyl in low concentrations did not unfold the protein [Aquaporin Expression Contributes to Human Transurothelial Permeability in vitro and Is Modulated by NaCl Rubenwolf, Peter C.; Georgopoulos, Nikolaos T.; Kirkwood, Lisa A.; Baker, Simon C.; Southgate, Jennifer; Ko, Ben C. B.]. 158 159 160 Figure S6 Mbp-dHBx sequencing by MALDI-TOF 1250 ENLYFQGLNLDASYLTQPLFATNVIR MW theoretical: 299 MW measured fits to GLNLDASYLTQPLFATNVIR I/L V N T A F Q T I/L Y S A 1000 750 500 250 161 162 163 164 165 166 167 168 0 250 500 750 1000 1250 1500 1750 2000 m/z (ENLYFQG) = TEV protease cleavage site Starting from (L) begins the sequence of the DHBx The dhbx protein was identified by exctracting the incomplete cleaved band from an SDS- PAGEl applying Matrix Laser Desorption Ionization Time of Flight (MALDI-TOF) mass spectrometry. The (M+H) + values for the peptide fragments, produced by trypsin digestion, were used for protein identification applying the MASCOT-MS software. The sequences identified match pars of the sequence of dhbx. 169 170 171 172 173 174 175 176
Supporting information, sup-14 177 178 Figure S7 SDS PAGE of the Mbp-dHBx complex M 1 2 3 4 5 6 7 8 116.0 66.2 45.0 35.0 25.0 179 180 181 182 183 184 185 186 187 188 189 190 M = Marker 1 = Fusion protein MBP-dHBx (control). 2 = Dialyzed fusion protein MBP-dHBx (Maltose removal). 3 = Dialyzed fusion protein MBP-dHBx with Tridecyl-β-D-maltoside detergent (application 1:1) 4 = Flow through fraction of the dialyzed fusion protein using a affinity column. 5 = Washing fraction (1) of the dialyzed fusion protein using a affinity column. 6 = Washing fraction (2) of the dialyzed fusion protein using a affinity column. 7 = Elution fraction (1) of the dialyzed fusion protein using a affinity column. 8 = Elution fraction (2) of the dialyzed fusion protein using a affinity column.