Nature Methods: doi: /nmeth Supplementary Figure 1

Transcription

1 Supplementary Figure 1 Subtiligase-catalyzed ligations with ubiquitin thioesters and 10-mer biotinylated peptides. (a) General scheme for ligations between ubiquitin thioesters and 10-mer, biotinylated peptides. (b) Representative SDS-PAGE analysis of the purification of an ubiquitin-mesna thioester. MW, molecular weight markers; T, total cell pellet; L, E. coli cell lysate; R, chitin resin; E1 and E2, elutions from chitin resin. (c) SDS-PAGE analysis of subtiligase treatment of recombinant ubiquitin thioester (top) or standard ubiquitin (bottom) with peptide 1 (P 1 P 2 = GL). MW, molecular weight markers; N, no subtiligase control. (d) SDS- PAGE analysis of subtiligase-catalyzed ligations between Ubiquitin-MESNA (P 4 P 1 = LY) and the indicated peptides (peptides 2-4, and 6-10, Supp. Table 3). Representative of two replicates as reported in Supplemental Table 1. (e) SDS-PAGE analysis of subtiligasecatalyzed ligations between the indicated Ubiquitin-MESNA construct and peptide 1 (P 1 P 2 = GL). Representative of two replicates as reported in Supplemental Table 1.

2 Supplementary Figure 2 Mass spectrometry analysis of the ligation product of ubiquitin and peptide 1 (P 1 P 2 = GL). (a) MALDI-MS measurement of ligation product of G76Y ubiquitin (P 4 P 1 = LY) and peptide 1 (P 1 P 2 = GL) purified by mono-avidin beads via a C-terminal biotin on peptide 1 (Supplemental Table 3). (b) Purification of ligation product by mono-avidin agarose chromatography. MW, molecular weight markers, Lane 1, ligation reaction before loaded on mono-avidin agarose; lane 2, flow-through from mono-avidin agarose. The agarose was washed with PBS buffer and the biotinylated ligation product was eluted with 2 mm biotin in PBS buffer. The eluate with 2.5 µg of ligation product was subjected to trypsin digestion with 50 ng trypsin in 50 mm Tris-HCl, ph 8.0 at 37 o C for 3 hrs and stopped by 0.1% TFA, and analyzed by MALDI-MS. (c) MALDI-MS measurement of tryptic digestion with the mass of the fragment containing the ligation site highlighted. MS data was analyzed using the Protein Analysis Worksheet (Genomic Solutions) as shown in (d) demonstrating excellent correspondence between the calculated and observed masses for the peptide fragment containing the ligation site (red box).

3 Supplementary Figure 3 Analysis of subtiligase-mediated ubiquitin thioester hydrolysis. Ubiquitin-MESNA was incubated with subtiligase for 5 minutes at 25 o C and then quenched with 1% TFA in water prior to MALDI-MS analysis. A negative control without subtiligase is included for comparison. (a) MALDI-MS measurements for Ubiquitin-MESNA with (right) or without (left) subtiligase treatment. (b) Schematic showing proposed partitioning of the acylated enzyme intermediate between aminolysis and hydrolysis.

4 Supplementary Figure 4 Generation of Y217K subtiligase to improve the efficiency of ligations with acidic residues at the ligation junction. Y217K subtiligase was designed and prepared based on structural studies on subtilisin 1 and its potential to complement acidic residues at the P1 position. (a) Introducing the Y217K mutation in the active site of subtilisin. The catalytic triad Ser/Cys221, His64 and Asp32, as well as Pro225 are highlighted. Left: crystal structure of subtilisin complexed with a chymotrypsin inhibitor (pink) containing a Glu residue in the P1 site (adapted from PDB: 2SNI 2 ). Right: crystal structure of S221C, Y217K subtilisin with the sidechain of Lys217 oriented toward His64 and potentially complementary to acidic residues in the P1 site (adapted from PDB: 1SUD 3 ). (b) Coomassiestained SDS-PAGE of purified subtiligase variants. MW, molecular weight markers. (c) SDS-PAGE analysis of ligations between Ubiquitin-MESNA (P 4 P 1 = LE) and peptides 1 and 5 (Supplemental Table 3). (d) SDS-PAGE analysis of ligations between Ubiquitin- MESNA (P 4 P 1 = LD) and peptides 1 and 5. Abbreviations used: Std, standard subtiligase; QK, E156Q-G166K subtiligase; Y217K, Y217K subtiligase. 1. Pantoliano, M. W. et al. Biochemistry 28, (1989). 2. McPhalen, C. A. & James, M. N. Biochemistry 27, (1988). 3. Gallagher, T., Bryan, P. & Gilliland, G. L. Proteins 16, (1993).

5 Supplementary Figure 5 Comparative kinetics between native chemical ligation and subtiligase-catalyzed ligation. (a) Native chemical ligation. (b) Subtiligase-catalyzed ligation. MW, molecular weight markers. Each ligation was run twice and the span of values is shown (c). Each reaction contained 100 µm ubiquitin thioester (P 4 P 1 = LY) and 3 mm peptide. Peptides 11 (P 1 P 2 = CR) and 1 (P 1 P 2 = GL) were used for (a) and (b), respectively (Supplemental Table 3). 0.5 µm subtiligase was used for the subtiligasecatalyzed ligation. For native chemical ligation, the reaction was quenched by 50 mm cysteine on ice for 20 minutes and the excess of cysteine was removed by trichloroacetic acid precipitation.

6 Supplementary Figure 6 Subtiligase-catalyzed ligations with GST-thioesters and 10-mer biotinylated peptides. (a) General scheme for ligations between GST-thioesters and 10-mer, biotinylated peptides. (b) Representative SDS-PAGE analysis of the purification of a GST-MESNA thioester. MW, molecular weight markers; L, E. coli cell lysate; FT, flow-through after loading lysate onto chitin resin; R, chitin resin; E, elution from chitin resin; C, concentrated GST-MESNA. (c) SDS-PAGE analysis of all GSTthioesters used for ligations. MW, molecular weight markers. (d) Western blot analysis of subtiligase-catalyzed ligations between the indicated GST-MESNA thioester and peptides 1-3 (Supplemental Table 3). Representative of two replicates as reported in Supplemental Table 2.

7 Supplementary Figure 7 Purification of r-pten-mesna and semisynthetic PTEN. (a) Representative SDS-PAGE analysis of the purification of r-pten-mesna. MW, molecular weight markers; L, insect cell lysate; C, lysate after cellulose treatment; FT, flow-through after loading lysate onto chitin resin; E, elution from chitin resin. (b) Representative size-exclusion chromatogram and SDS-PAGE analysis of PTEN ligation. MW, molecular weight markers. (c) Representative SDS- PAGE analysis of monoavidin purification of semisynthetic PTEN. MW, molecular weight markers; Lane 1, 0.5 M NaCl wash; Lane 2, 1 M NaCl wash; Lane 3, 150 mm NaCl rinse; Lanes 4-7, 10 mm biotin elution; Lane 8, r-pten-mesna. (d) SDS-PAGE gel showing purified PTEN variants. MW, molecular weight markers. (e) Anti-phosphorylated-PTEN antibodies respond more strongly to Y379-4p- PTEN than to C379-4p-PTEN. Each image is normalized to the C379-4p-PTEN band.

8 Supplementary Figure 8 MALDI-MS analyses of peptides used in this work. (a-l) Mass spectra of the indicated peptides.

9 Protocol for Enzyme Catalyzed Expressed Protein Ligation 1. Subclone the cdna for a protein fragment of interest N-terminal to an intein-cbd-containing vector (available from New England BioLabs). In the first and fourth residues upstream of the intein, in the protein fragment, avoid acidic residues and where possible include hydrophobic residues. 2. Express the fusion protein in the appropriate heterologous host (e.g. E. coli or baculovirus-sf9/high Five insect cells). 3. Lyse the cells by an appropriate method. a. If insect cells were used, incubate the lysate with powdered cellulose to remove chitinase (about 1 ml cellulose for every 2 ml lysate). 4. Immobilize the fusion protein fusion on chitin resin and wash to remove impurities. 5. Treat the immobilized fusion protein with cleavage buffer containing 250 mm NaCl, 50 mm HEPES, 1 mm EDTA, ph = 7.5, and 300 mm MESNA, overnight and isolate the protein thioester in the eluate. 6. Exchange the protein thioester (by dialysis or ultrafiltration, as needed) into a buffer containing 150 mm NaCl, 50 mm MES, ph ph 6 is preferred to avoid hydrolysis of the thioester, but up to 6.5 can be used if the protein of interest cannot tolerate a lower ph. 7. Concentrate the protein to 1 mg/ml or greater. If not used within a day, flash-freeze for storage. 8. Mix the protein thioester (0.4 mg/ml or greater) with synthetic peptide (1-10 mm) and subtiligase ( µm) in reaction buffer containing 100 mm bicine, ph 8, mm CaCl 2 at 25 C 9. After 5-90 minutes, analyze the reaction mixture using SDS-PAGE to determine degree of ligation. a. For challenging ligations, use greater concentrations of peptide and/or subtiligase, and longer reaction times. For peptide/proteins with acidic residues at the ligation junction use E156Q,G166K or Y217K subtiligase. For peptides containing phosphates, use a highcalcium buffer 10. Once the ligation is optimized, purify the ligated protein using chromatography appropriate for the protein of interest.

10 Supplementary Tables Ubiquitin thioester (P4 P1) Leu-Arg-Gly-Tyr Supp. Table 1 Peptide (P1 and P2 ) Percent Conversion Gly-Gly 47 ± 1 Ala-Leu 55 ± 2 Pro-Leu 9 ± 1 Phe-Leu 61 ± 1 Met-Thr 50 ± 1 Tyr-Leu 60 ± 2 Ser-Ile 58 ± 1 Arg-Leu 63 ± 2 Glu-Leu 20 ± 2* Gly-Leu 69 ± 2 Ala-Arg-Gly-Tyr 56 ± 3 Leu-Arg-Gly-Ala 47 ± 6 Leu-Arg-Gly-Val 63 ± 6 Leu-Arg-Gly-Leu 61 ± 3 Leu-Arg-Gly-Ile 62 ± 2 Leu-Arg-Gly-Phe Gly-Leu 55 ± 3 Leu-Arg-Gly-Tyr 69 ± 2 Leu-Arg-Gly-Trp 63 ± 0.1 Leu-Arg-Gly-His 56 ± 3 Leu-Arg-Gly-Glu 48 ± 1 Glu-Arg-Gly-Tyr < 5 Ubiquitin thioester ligations catalyzed by subtiligase. Reactions were carried out as described in the Methods. Percent conversions are averages of two replicates based on quantifying Coomassie-stained SDS-PAGE and errors represent the span of duplicates performed on separate occasions. *Performed with Y217K subtiligase.

11 GST thioester (P 4 - P 1 ) Supp. Table 2 Peptide (P 1 and P 2 ) Gly-Leu Met-Thr Ser-Ile Percent Converted Phe-Ala-Ala-Tyr 53 ± 3 40 ± 3 42 ± 0.4 Phe-Ala-Ala-Thr 53 ± 3 49 ± 6 49 ± 4 Phe-Ala-Ala-Arg 52 ± 4 35 ± 3 41 ± 5 Phe-Ala-Ala-Gly 35 ± ± 5 36 ± 6 Ser-Ala-Ala-Tyr 61 ± 3 56 ± 4 64 ± 10 Ser-Ala-Ala-Gly < 5 < 5 < 5 Asp-Ala-Ala-Tyr < 5 < 5 < 5 Phe-Ala-Ala-Glu 63 ± 1 58 ± 2 59 ± 4 GST thioester ligations catalyzed by subtiligase. Reactions were carried out as described in the Methods. Percent conversions are averages of two replicates based on quantifying western blots by using a biotin tag in the peptides and errors represent the span of duplicates performed on separate occasions.

12 Supp. Table 3 ID Sequence 1 GLSGRGKGGK-Biotin 2 MTSGRGKGGK-Biotin 3 SISGRGKGGK-Biotin 4 ALSGRGKGGK-Biotin 5 ELSGRGKGGK-Biotin 6 RLSGRGKGGK-Biotin 7 PLSGRGKGGK-Biotin 8 YLSGRGKGGK-Biotin 9 GGSGRGKGGK-Biotin 10 FLSGRGKGGK-Biotin 11 CRGKGGKGLGKGGAK-Biotin 4p-tail RYpSDpTpTDpSDPENEPFDEDQHTQITK-Biotin np-tail RYSDTTDSDPENEPFDEDQHTQITK-Biotin Synthetic peptides used in this study.