Supplementary Results Supplementary Table 1. P1 and P2 enrichment scores for wild-type subtiligase.
Supplementary Table 2. Masses of subtiligase mutants measured by LC-MS.
Supplementary Table 2 (cont d). Masses of subtiligase mutants measured by LC-MS.
Supplementary Table 3. GFP and GFP variant masses measured by LC-MS.
Supplementary Table 4. Dissociation constants for modified recombinant antibodies.
Supplementary Table 5. Two-tailed, unpaired t-test results for signal peptidase N termini capture by stabiligase.
Supplementary Table 6. Two-tailed, unpaired t-test results for signal peptidase N termini capture by the cocktail of four stabiligase mutants.
Supplementary Table 7. List of oligonucleotides used for plasmid construction and sitedirected mutagenesis.
Supplementary Table 7 (cont d). List of oligonucleotides used for plasmid construction and site-directed mutagenesis.
Supplementary Table 8. List of supplementary dataset information and ProteomeXchange Accession Numbers.
Supplementary Table 8 (cont d).
Supplementary Table 8 (cont d).
Supplementary Figure 1. IceLogos for proteome-derived peptide libraries. (a) IceLogo for tryptic peptide acceptor library derived from the E. coli proteome. Amino acids that are enriched relative to natural abundance are shown above the line and amino acids that are de-enriched are shown below the line. (b) IceLogo for GluC peptide library derived from the E. coli proteome. IceLogos were generated using the IceLogo server 1 by using the first five amino acids of each peptide identified in the library as the experimental set and the pre-compiled Swiss-Prot composition for E. coli DH10B as the reference set. The scoring system was fold change with a p value of 0.05.
Supplementary Figure 2. Chemical structure of biotinylated subtiligase substrate. The subtiligase substrate, biotin-eeenlyfq-glycolate-r-nh 2, includes a biotin tag for affinity purification (green), a TEV protease cleavage sequence (ENLYFQ) for selective elution of subtiligated peptides (red), an aminobutyric acid (Abu) tag immediately following the TEV protease site, and a glycolate ester subtiligase acylation site (blue).
Supplementary Figure 3. PILS specificity maps for subtiligase alanine scan mutants. Sequences that were enriched compared to the input library are colored in blue and sequences that were de-enriched compared to the input library are colored in red.
Supplementary Figure 3 (cont d). PILS specificity maps for subtiligase alanine scan mutants.
Supplementary Figure 4. Kinetic analysis of subtiligase alanine mutants. a) Design of FRET assay for subtiligase-catalyzed ligation and ester hydrolysis. The blue star represents the Pacific Blue fluorophore (ex = 410 nm, em = 455 nm), the green star represents a 5-/6- carboxyfluorescein (FAM) fluorophore (ex = 495 nm, em = 520 nm), and the quencher represents Dabcyl. (b) Fluorescence spectra of hydrolysis product (top left), ligation product and a variant lacking Pacific Blue (top right), quenched substrate in the presence or absence of subtiligase (bottom left), and quenched substrate and FAM-labeled nucleophile in the presence or absence of subtiligase. (c) Workflow for kinetics measurements. (d) Relative k cat /K M values for subtiligase mutants compared to wild-type subtiligase.
Supplementary Figure 5. Structural analysis of class I subtiligase mutants. Structure of the subtilisin-ssi complex 2 showing the P4-P1 residues of SSI in purple and the P1'- P2' residues of SSI in teal. The sites of class I alanine mutants are shown in light blue and the site of class II mutations are shown in light green. Positions of class I and class II mutations are labeled in black. Sites of other alanine mutations are shown in grey.
Supplementary Figure 6. PILS specificity maps for Y217 and F189 mutants. Sequences that were enriched compared to the input library are colored in blue and sequences that were deenriched compared to the input library are colored in red.
Supplementary Figure 6 (cont d). PILS specificity maps for Y217 and F189 mutants.
Supplementary Figure 6 (cont d). PILS specificity maps for Y217 and F189 mutants.
Supplementary Figure 7. HPLC analysis of product ratio for subtiligase-m222a. Reaction mixtures containing 1 M subtiligase variant, 350 M suc-aapfglcfg-nh 2 (ester substrate) and 350 M AF-NH 2 (nucleophile substrate) were incubated at room temperature for 1 h and analyzed by HPLC. (a) HPLC chromatograms for wild-type subtiligase and subtiligase-m222a. (b) Quantification of relative hydrolysis product and ligation product peak areas.
Supplementary Figure 8. PILS specificity maps for additional subtiligase mutants. Sequences that were enriched compared to the input library are colored in blue and sequences that were de-enriched compared to the input library are colored in red.
Supplementary Figure 8 (cont d). PILS specificity maps for additional subtiligase mutants.
Supplementary Figure 9. Subtiligase-catalyzed modification of native E. coli proteins. (a) E. coli lysate assay to examine the scope of native proteins that can be labeled by subtiligase and variants. Lysates were prepared under native conditions and labeling with stabiligase, stabiligase-m222a, stabiligase-y217k/m222a, or stabiligase-f189r/m222a and biotinylated ester 1. (b) Weighted Venn diagram showing the overlap in N-termini labeled by each enzyme and 50% expansion of native proteins ligated by the new subtiligase variants.
Supplementary Figure 10. Subtiligase mutant specificity for native E. coli proteins. The set of N-terminal peptides identify using wild-type stabiligase for enrichment was used as the reference set. Sequences that were enriched compared to wild-type stabiligase are colored in blue and sequences that were de-enriched compared to wild-type stabiligase are colored in red.
Supplementary Figure 11. Subtiligase and mutant labeling of recombinant antibodies with extended N termini. An anti-gfp recombinant antibody ( GFP rab) with the light chain N terminus extended by zero, one (Gly), two (Gly-Gly), three (Gly-Gly-Gly), or four (Gly-Gly- Gly-Ser) residues was labeled with ester 2 (N 3 AAPF-glycolyate-FG) and stabiligase-m222a or stabiligase-f189r/m222a. Modification yields of 11% (native N terminus), 21% (Gly), 32% (Gly-Gly), 53% (Gly-Gly-Gly), and 62% (Gly-Gly-Gly-Ser) were observed, suggesting that N- terminal accessibility impacts modification yield.
Supplementary Figure 12. Subtiligase and mutant labeling of recombinant antibodies with orthogonal light chain and heavy chain N termini. An anti-gfp recombinant antibody ( GFP rab) was labeled with ester 2 (N 3 AAPF-glycolyate-FG) and subtiligase-y217k or stabiligase. (a) For the unmodified rab sequence, no labeling was observed with stabiligase, while quantitative labeling was observed with subtiligase-y217k. (b) When an AFA extension was added to the N-terminus of the light chain, quantitative labeling of the light chain was observed with stabiligase, while quantitative labeling of both the light and heavy chains was observed with subtiligase-y217k.
Supplementary Figure 13. Protein A labeling with a panel of subtiligase mutants. Protein A (50 M) was labeled with 5 mm ester 2 (N 3 AAPF-glycolyate-FG) and 1 M subtiligase variant and analyzed by mass spectrometry to determine the extent of labeling. 2x indicates that the protein A reaction mixture was desalted and labeled a second time.
Supplementary Figure 14. Characterization of synthetic peptides. Strategy for one-step, subtiligase-catalyzed protein modification. Peptide ester 2 reacts with commercially available NHS esters, providing a convenient route to site-specific labeling reagents.
Supplementary Figure 15. Subtiligase DNA and protein sequences. (a) Codon-optimized subtiligase sequence used for protein expression. (b) Protein sequence of mature subtiligase following autoproteolysis to remove the pro domain. a DNA sequence of synthetic gene: GTGAGAGGCAAAAAAGTATGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGGCGTTCGGCA GCACATCCTCTGCCCAGGCGGCCGGTAAATCCAACGGTGAGAAAAAATATATTGTAGGCTTCAAACAAAC CATGAGCACCATGTCGGCTGCCAAAAAAAAAGACGTCATTTCAGAGAAGGGTGGGAAGGTACAAAAACAG TTCAAATATGTAGATGCGGCCTCCGCCACGTTGAACGAAAAGGCGGTAAAAGAACTGAAAAAAGATCCGT CAGTGGCATACGTAGAAGAAGATCATGTCGCGCATGCTTATGCTCAAAGCGTCCCGTACGGCGTCTCACA GATCAAGGCACCGGCGCTGCACAGCCAGGGTTATACCGGCTCCAACGTTAAGGTGGCGGTCATTGATAGC GGCATCGATAGCTCCCATCCCGACCTCAAAGTTGCCGGCGGCGCTTCTATGGTGCCAAGCGAAACTAATC CTTTTCAGGATAATAATAGTCACGGGACGCATGTAGCAGGTACAGTCGCCGCTTTGAATAATTCTATCGG CGTGCTGGGTGTTGCGCCGAGCGCGTCACTCTACGCCGTGAAAGTGCTGGGCGCGGACGGCAGCGGACAA TATAGTTGGATTATTAATGGCATCGAGTGGGCCATCGCGAACAATATGGATGTGATCAATATGAGCCTGG GCGGCCCAAGCGGCAGTGCTGCCTTAAAAGCGGCGGTGGATAAAGCTGTGGCAAGTGGGGTCGTCGTGGT GGCAGCGGCGGGCAATGAAGGCACGAGTGGCTCTTCTTCGACTGTCGGATACCCCGGCAAATACCCGTCG GTCATCGCGGTTGGGGCGGTTGATAGCTCTAACCAACGTGCCAGTTTTAGCAGTGTAGGCCCAGAATTAG ATGTGATGGCGCCAGGTGTGTCTATCCAGAGCACACTCCCGGGCAATAAATATGGTGCGTATAATGGCAC ATGTATGGCCAGTGCGCACGTTGCCGGGGCGGCGGCCCTGATCTTAAGTAAACATCCAAACTGGACCAAC ACCCAGGTGCGTAGCAGTTTGGAAAACACCACCACGAAACTGGGTGATTCTTTTTATTACGGGAAAGGTC TCATCAATGTTCAAGCGGCCGCCCAACTCGAGCACCACCACCACCACCACTAA b Protein sequence of mature subtiligase: AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASFVPSETNPFQDNNSHGTHVAG TVAALDNSIGVLGVAPSASLYAVKVLGADGSGQYSWIISGIEWAIANNMDVINLALGGPSGSAALKAAVD KAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLP GNRYGAYSGTCMASAHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQLEHHH HHH Supplementary Figure 16. Characterization of synthetic peptides. Synthetic peptides were
analyzed by LC-MS. Evaporative light scattering chromatograms are shown and peaks are labeled with the measured m/z values. (b) Biotin-EEENLYFQ-Abu-glc-R-NH 2. (b) N 3 Ac- AAPC-glc-FG-NH 2. (c) Suc-KAAPF-glc-FG-NH 2. (d) NHS-biotin-modified suc-kaapf-glc- FG-NH 2. Ac, acetyl; Suc, succinyl. Literature Cited
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