Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials

Transcription

1 Supporting information Volume 71 (2015) Supporting information for article: 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

2 Supporting information, sup 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-C12E Octaethylene glycol monododecyl ether ANAPOE-C12E ANAPOE-C13E n-dodecyl-β-d-maltopyranoside n-dodecyl-α-d-maltopyranoside CYMAL ANAPOE-X ANAPOE-C12E n-undecyl-β-d-thiomaltopyranoside ANAPOE-X Sucrose monododecanoate CYMAL n-undecyl-α-d-maltopyranoside / / / / / / / / / / / / / / / / / / / / / / / / / / / /- 52.1

3 Supporting information, sup-2 15 n-undecyl-β-d-maltopyranoside CYCLOFOS Pentaethylene glycol monodecyl ether n-decyl-α-d-maltopyranoside ANAPOE-C10E / / / / / / / / / / n-dodecyl-n,n-dimethylamine-n-oxide (LDAO or DDAO) / / ANAPOE-C10E n-decyl-β-d-maltopyranoside CYMAL n-nonyl-β-d-thiomaltopyranoside Dimethyldecylphosphine oxide n-nonyl-β-d-maltopyranoside n-nonyl-β-d-glucopyranoside CYMAL Tetraatheylene glycol monooctyl ether n-octyl-β-d-thiomaltopyranoside Hexaethylene glcol monooctyl ether (C8E6) / / / / / / / / / / / / / / / / / / / / / / ANAMEG / /

4 Supporting information, sup 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).

5 Supporting information, sup

6 Supporting information, sup

7 Supporting information, sup

8 Supporting information, sup 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 % 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 g. E: Step 5: BR at 2 mg/ml in 2% Triton X-100 in 20 mm sodium acetate, centrifuged for 35 min at g. F: Step 6: BR at 2 mg/ml in 2% Triton X-100 in 20 mm sodium acetate, centrifuged 20 min at g

9 Supporting information, sup

10 Supporting information, sup-9 83 Figure S3 SDS-PAGE von BR obtained after individual purifications steps 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

11 Supporting information, sup Figure S4 Reference DLS measurement of pure Triton X-100 in 20 mm sodium acetate at ph

12 Supporting information, sup 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)

13 Supporting information, sup 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 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 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

14 Intens. [a.u.] Acta Cryst. (2015). F71, doi: /s x Supporting information, sup 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.] 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 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

15 Supporting information, sup Figure S7 SDS PAGE of the Mbp-dHBx complex M 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.