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1 1 upplementary Information Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic prenylated protein probes Yao-Wen Wu*,, Lena K. esterlin, Kui-Thong Tan, erbert Waldmann, Kirill Alexandrov and Roger. Goody*, Affiliation: Department of Physical Biochemistry and Department of Chemical Biology, Max Planck Institute of Molecular Physiology, tto-ahn-trasse 11, Dortmund, Germany; Institute for Molecular Bioscience and Australian Institute for Bioengineering and anotechnology, University of Queensland, Brisbane, Queensland, Australia * Corresponding authors: Roger Goody; Fax: ; Yaowen Wu; Fax: ; goody@mpi-dortmund.mpg.de yaowen.wu@mpi-dortmund.mpg.de 1

2 2 upplementary Methods Preparation of Rab1-BD-farnesyl Rab1b for enzymatic prenylation was C-terminally modified (CC replaced by CVIL motif) and codon optimized for expression in E. coli. The protein was produced as an -terminal fusion with a 6is-MBP(Maltose Binding Protein) tag in E. coli BL21(DE3). Purification of Rab1CVIL was achieved using a combination of i-ida chromatography, proteolytic removal of the MBP tag, gel filtration and amylose affinity chromatography and the protein was stored in buffer containing 20 mm epes p 8, 50 mm acl, 2 mm DTE, 1 mm MgCl 2 and 10 µm GDP. BD-farnesyl pyrophosphate (BD-FPP) was produced as described elsewhere 1. 6is-GT- GGTase-I and GGTaseI were coexpressed in E. coli and copurified with a combination of i- IDA chromatography and gelfiltration. To produce Rab1-F, Rab1CVIL was incubated for 1 hour at 25 o C with recombinant 6is-GT-GGTase-I and BD-FPP in a molar ratio of 1:0.5:5. GGTase-I was separated from Rab1-F using i-ida chromatography. 2

3 3 uppelmentary Results Figure 1. ptimization of the reaction conditions for the ligation of Rab7 3-MEA with Cys(tBu)erCys(BD-farnesyl). (a) creening of detergent for ligation. Reaction was run overnight at 30 C in the presence of 50 mm detergent. (b) Ligation in the presence of various concentrations of CTAB at 30 C. Figure 2. The ligation of Rab7 3-MEA with Cys(tBu)erCys(BD-farnesyl). (a) The structure of CTAB. (b) Ligation in the presence of 40 mm CTAB at different temperatures. As a control Rab7wt was present in the reaction instead of Rab7 3-MEA thioester. The lower columns in each panel show the quantification of the corresponding fluorescent bands. 3

4 4 Figure 3. Purification of BD-farnesylated Rab7. (a) After reaction, supernatant () and pellet (P) were separated. In the control experiments, Rab7wt was used instead of Rab7 3-MEA thioester in the presence of peptide and the absence of peptide. (b) Quantification of the fluorescence of the protein bands shown in a. (c) eparation of protein and unligated peptide by washing with organic solvents. T: mixture after reaction, : supernatant after reaction, W: Me solution after washing the pellet, P: pellet after washing. (d) Quantification of the fluorescence of the corresponding protein and peptide bands shown in c. Figure 4. Gel filtration chromatography and EI-M of Rab7-F. (a) Gel filtration profile of Gppp:Rab7-F using a uperdex-200 column (upper panel). The dashed line represents an 4

5 5 elution profile detected by the absorbance at 280 nm while the solid line represents detection of BD fluorescence. The resulting fractions are resolved by D-PAGE as shown by Coomassie blue staining (middle panel) and fluorescence scan (lower panel). The elution position of molecular weight standards is shown as arrows in the upper panel. (b) Electrospray ionisation-mass spectrometric (EI-M) analysis of Rab7-F (M calc = 23,770 Da). Figure 5. The emission spectra of Rab7-F before (solid line) and after binding to REP-1 and GDI-1 (dashed line). (a) GDP:Rab7-F binds to REP-1. (b) GDP-Rab7-F binds to GDI-1. (d) Gppp:Rab7-F binds to REP-1. (e) The emission spectra of 100 nm Gppp:Rab7- F in the presence of 1.05 M GDI-1 (red line), 2.1 M GDI-1 (blue line) and 2.7 M GDI-1 (green line). Excitation was set to 479 nm. (c) Quantitative analysis of GDP:Rab7-F interaction with GDI-1. Titration of GDI-1 to 10 nm GDP:Rab7-F. K d values were obtained by fitting the data to a quadratic equation shown in the experimental section. Figure 6. Gel filtration chromatography of GDP- (A) and Gppp-bound (B) Rab7-F in complex with GDI-1. The upper panel shows the elution profile using a uperdex-200 column. 5

6 6 The resulting fractions were analyzed by D-PAGE and a fluorescence scan was performed (lower panel). Figure 7. The emission spectra of GDP:Rab7BD-G (a, b) and Gppp:Rab7BD-G (c, d) in the absence (black line) and the presence (red line) of REP-1 (a, c) or GDI-1 (b, d), λ ex =479nm. 6

7 7 Figure 8. Interaction of diprenylated Rab7 with REP-1 and GDI-1. (a, d) The emission spectra of Rab7dans-GG in the absence (solid line) and the presence (dashed line) of REP-1 (a) and GDI-1 (d), with the excitation at 340nm. (b, e) The emission spectra of Rab7dans-GG in the absence (solid line) and the presence (dashed line) of REP-1 (b) and GDI-1 (e), with the excitation at 280nm. The changes in the signal of fluorescence resonance energy transfer (FRET) from Trp to dansyl (480 nm) can be observed when Rab7dans-GG binds to REP-1 and GDI-1. (c, f) Titration of REP-1 (c) to 50nM GDP:Rab7dans-GG and GDI-1 (f) to 30nM GDP:Rab7d-GG. (λ ex/em :340/479nm). K d values were obtained by fitting the data to a quadratic equation. Figure 9. The emission spectra of 120 nm Gppp:Rab7dans-GG in the presence of 1.13 M REP-1 (red line), 1.7 M REP-1 (green line) and 2.26 M REP-1 (blue line). Excitation was set to 340 nm (a) and 280 nm (b). 7

8 8 Figure 10. Gel filtration chromatography of GDP- (a, c) and Gppp-bound (b, d) Rab7dans-GG in complex with REP-1 (a, b) and GDI-1 (c, d). The upper panel shows the elution profile using a uperdex-200 column. The black line represents an elution profile detected by absorbance at 280 nm while the red line represents detection of dansyl fluorescence. The elution position of molecular weight standards is shown as arrows. The resulting fractions were analyzed by D-PAGE (fluorescence scan:middle panel; Coomassie blue staining: lower panel). 8

9 9 Figure 11. Dissociation of 60 nm GDP:Rab7-F from 60 nm REP-1 (a) and 60 nm GDI-1 (c). Arrows indicate the time of addition of 600 nm of the displacer mono-geranylgeranylated Rab7 complexed with GGT. (b) 32 nm Gppp:Rab7-F in complex with 300 nm REP-1 complex was rapidly mixed with 2 M GDP:Rab7 on a stopped-flow machine to displace Gppp:Rab7-F. (d) 25 nm Gppp:Rab7BD-G in complex with 240 nm REP-1 was dissociated by 3.3 M GDP:Rab7. The fluorescent decay upon dissociation of Rab7-F from REP-1 or GDI-1 was recorded using BD fluorescence. The red lines show the fit of the traces to single exponential functions. 9

10 10 Figure 12. Dissociation of 50 nm GDP:Rab1-F from 50 nm GDI-1. At the time point indicated by the arrow, 200 nm mono-geranylgeranylated Rab7 complexed with GGT was added. The red lines show the fit of the traces to a single exponential function. Table 1. bserved dissociation rate constants of GDP-Rab1-F:GDI-1 complex under the influence of DrrA in the presence and the absence of GTP. DrrA (µm) k obs (s -1 ) without GTP k obs (s -1 ) with 100 µm GTP Characterization of peptides C(tBu)C(BD-farnesyl) 2 2 The desired product (97 %) was found as dark red powder. 1 MR (400 Mz, CD 3 D): δ = 8.50 (d, J = 8.9 z, 1), 6.28 (dd, J = 8.9, 0.8 z, 1), (m, 1), (m, 1), (m, 1), (m, 1), 4.16 (dd, J = 8.6, 4.9 z, 1), (m, 2), 3.83 (dd, J = 7.5, 5.6 z, 2), (m, 1), 3.13 (dd, J = 3.3, 1.6 z, 1), 3.06 (dd, J = 14.4, 8.7 z, 1), 2.99 (dd, J = 14.0, 4.6 z, 1), 2.78 (dd, J = 13.7, 7.8 z, 1), (m, 2), (m, 8), 1.70 (s, 3), 1.63 (s, 3), 1.57 (s, 3), 1.36 (s, 9) ppm. LC-M (C4) [M+] +, [M+a] + ; R t = 7.50 min. RM (EI) m/z calc. for C , found [M+] + C(tBu)K(BD)C(tBu)C(G)-Me The desired product (95 %) was found as yellowish-green oil. R f : 0.50 (C 2 Cl 2 /Me, 10:1, v/v). 1 -MR (400 Mz, CDCl 3 /CD 3 D 10:1, v/v): = 8.51 (d, 3 J = 8.4 z, 1, C-6 BD), 6.25 (d, 3 J = 8.8 z, 1, C-5 BD), 5.20 (t, 3 J = 7.4 z, 1, C GG), (m, 3, 3 * C GG), 4.72 (dd, 3 J 1 = 9.0 z, 3 J 2 = 4.6 z, 1, -C Cys(tBu)), 4.67 (dd, 3 J 1 = 7.4 z, 3 J 2 = 5.0 z, 1, -C Cys(GG)), 4.51 (dd, 3 J 1 = 3 J 2 = 4.8 z, 1, -C er), 4.45 (dd, 3 J 1 = 3 J 2 = 5.4 z, 1, -C er ), (m, 1, -C Lys), (m, 2, 2 * -

11 11 C 2a er), (m, 2, 2 * -C 2b er), 3.76 (s, 3, C3), 3.73 (dd, 3 J 1 = 8.0 z, 3 J 2 = 4.4 z, 1, -C -Cys), 3.53 (br, 2, -C 2 Lys), (m, 2, -C 2a Cys(tBu), - C 2a ), ( -C 2a Cys, -C2b), 3.09 (dd, 2 J = 13.4 z, 3 J = 9.2 z, 1, -C 2b Cys(tBu)), 2.97 (dd, 2 J = 14.4 z, 3 J = 5.2 z, 1, -C 2a Cys(GG)), 2.89 (dd, 2 J = 13.6 z, 3 J = 8.0 z, 1, -C 2b -Cys), 2.82 (dd, 2 J = 13.8 z, 3 J = 7.8 z, 1, -C 2b Cys(GG)), (m, 13, 6 * C 2 GG, -C 2a Lys), (m, 3, -C 2b Lys, -C 2 Lys), 1.68 (s, 6, 2 * C 3 ), 1.60 (s, 9, 3 * C 3 ), (m, 2, -C 2 ), 1.34 (s, 9, C(C 3 ) 3 ). EI-M (m/z): calc. for C , found [M+] + C(tBu)K(dans)C(G)C(G)-Me 2 The desired product (99 %) was found as pale yellowish-green oil. R f : 0.35 (C 2 Cl 2 /Me, 20:1, v/v). [ ] D 20 : (c = 0.22, C 2 Cl 2 /Me, 10:1, v/v). 1 -MR (400 Mz, CDCl 3 /CD 3 D 10:1, v/v): = 8.53 (d, 3 J = 8.4 z, 1, C-2 dansyl), 8.31 (d, 3 J = 8.8 z, 1, C-8 dansyl), 8.19 (dd, 3 J = 7.2 z, J = 1.6 z, 1, C-4 dansyl), 7.57 (dd, 3 J 1 = 8.8 z, 3J2 = 7.6 z, 1, C-7 dansyl), 7.53 (dd, 3 J 1 = 8.4 z, 3 J 2 = 7.2 z, 1, C-3 dansyl), 7.21 (dd, 3 J = 7.6 z, J = 0.4 z, 1, C-6 dansyl), 5.22 (t, 3 J = 8.6 z, 1, C GG), 5.19 (t, 3 J = 9.0 z, 1, C GG ), (m, 6, 6 * C GG), 4.66 (dd, 3 J 1 = 8.0 z, 3 J 2 = 5.2 z, 1, - C Cys(GG)), 4.59 (dd, 3 J 1 = 8.0 z, 3 J 2 = 6.0 z, 1, -C Cys(GG) ), 4.51 (dd, 3 J 1 = 3 J 2 = 5.0 z, 1, -C er), 4.42 (dd, 3 J 1 = 3 J 2 = 5.2 z, 1, -C er ), 4.24 (dd, 3 J 1 = 8.0 z, 3 J 2 = 6.0 z, 1, -C Lys), 3.92 (dd, 2 J = 11.6 z, 3 J = 3.6 z, 1, -C 2a er), 3.90 (dd, 2 J = 11.2 z, 3 J = 4.4 z, 1, -C2a er ), (m, 3, 2 * -C2b er, -C Cys(tBu)), 3.74 (s, 3, C 3 ), (m, 4, -C 2, -C 2a, -C2a Cys(tBu)), 3.11 (dd, 2 J = 13.4 z, 3 J = 7.0 z, 1, -C 2b ), 3.01 (dd, 2 J = 13.8 z, 3 J = 5.8 z, 1, -C 2a Cys(GG)), 2.94 (dd, 2 J = 13.8 z, 3 J = 5.4 z, 1, -C2a Cys(GG) ), 2.89 (s, 6, (C 3 ) 2 ), (m, 4, -C2 Lys, -C 2b Cys(GG), -C 2b Cys(tBu)), 2.80 (dd, 2 J = 14.0 z, 3 J = 7.6 z, 1, -C 2b Cys(GG) ), (m, 24, 12 * C 2 GG), (m, 2, -C 2 Lys), (4 * s, 12, 4 * C 3 ), 1.60 (s, 18, 6 * C 3 ), (m, 4, -, -C 2 Lys), 1.35 (s, 9, C(C 3 ) 3 ). EI-M m/z: calcd for C , found (M+) +. Reference 1. Wu,Y.W., Alexandrov,K., & Brunsveld,L. ynthesis of a fluorescent analogue of geranylgeranyl pyrophosphate and its use in a high-throughput fluorometric assay for Rab geranylgeranyltransferase. at. Protoc. 2, (2007). 11