Thioamide-Based Fluorescent Protease Sensors

Transcription

1 Supporting Information Thioamide-Based Fluorescent Protease Sensors Jacob M. Goldberg, Xing Chen, Nataline Meinhardt, Doron C. Greenbaum, and E. James Petersson*, Department of Chemistry, University of Pennsylvania, 231 South 34 th Street, Philadelphia, PA USA Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, 433 South University Avenue, Philadelphia, Pennsylvania USA Current Address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA USA General Information. Boc-L-thionoleucine-1-(6-nitro)benzotriazolide, Boc-Lthionoalanine-1-(6-nitro)benzotriazolide, Boc-L-thionophenylalanine-1-(6-nitro)benzotriazolide, and Fmoc-β-(7-methoxycoumarin-4-yl)-Ala-OH were purchased from Bachem (Torrance, CA, USA). Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Leu- OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, and 2-chlorotrityl resin were purchased from Novabiochem (currently EMD Millipore; Billerica, MA, USA). Fmoc-D-Ala-OH was S1

2 (sequence: purchased from GenScript (Piscataway, NJ, USA). Piperidine and 2-(1H-benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from American Bioanalytical (Natick, MA, USA). Sigmacote, N,N-diisopropylethylamine (DIPEA), mouse serum, trypsin (type II from porcine pancreas; 1,000 2,000 units/mg dry solid), chymotrypsin (type II from bovine pancreas; 40 units/mg protein), thermolysin (from Bacillus thermoproteolyticus rokko; units/mg protein), papain (crude, from papaya latex; units/mg solid), and pepsin (from porcine gastric mucosa; 3,200 4,500 units/mg protein were purchased from Sigma-Aldrich (St. Louis, MO, USA). Native calpain-1 from human erythrocytes was purchased from Calbiochem (currently EMD Millipore). Calpastatin peptide Biotin-Asp-Pro-Met-Ser-Thr-Tyr-Ile-Glu-Glu-Leu-Gly-Lys-Arg-Glu-Val-Thr-Ile- Pro-Pro-Lys-Tyr-Arg-Glu-Leu-Leu-Ala-NH 2 ) was purchased from Auspep (Tullamarine, Victoria, Australia). All other reagents, including EZ-Link Sulfo-NHS-Biotin and immobilized neutravadin, were purchased from Fisher Scientific (Pittsburgh, PA, USA). Milli-Q filtered (18 MΩ) water was used for all solutions (EMD Millipore). Glass peptide synthesis reaction vessels (RVs) were treated with Sigmacote prior to use. UV absorbance spectra were obtained with a Hewlett-Packard 8452A diode array spectrophotometer (currently Agilent Technologies; Santa Clara, CA, USA). Fluorescence spectra were collected with a Tecan M1000 plate reader (San Jose, CA, USA), a Varian Cary Eclipse fluorescence spectrophotometer (currently Agilent Technologies), or a Photon Technologies International (PTI) QuantaMaster fluorometer (Birmingham, NJ, USA) fitted with a Peltier multicell holder. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) data were collected with a Bruker Ultraflex III MALDI-TOF-TOF mass spectrometer (Billerica, MA, USA). Peptides were purified with a Varian ProStar High-Performance Liquid Chromatography (HPLC) instrument outfitted with a S2

3 diode array detector (currently Agilent Technologies) using aqueous (H 2 O + 0.1% CF 3 CO 2 H) and organic (CH 3 CN + 0.1% CF 3 CO 2 H) phases. Peptide Synthesis and Purification. The peptides A'AFAµ, AAFAµ, L'LKAAµ, LLKAAµ, AKGLAAFAµ, AKGL'AAFAµ, L'PLFAERµ, LPLFAERµ, and F'aa (where µ is β- (7-methoxycoumarin-4-yl)-Ala; A', F' and L' denote thioalanine, thiophenylalanine, and thioleucine, respectively; and a denotes D-alanine) were each synthesized on a 12.5 µmol scale on 2-chlorotrityl resin. Since the first several amino acid couplings in each pair of oxo- and thiopeptides are identical, these residues were coupled on a 25 µmol scale in a common pot. The resin was then divided into two equal portions for the final amino acid coupling of either Leu or Leu' or Ala or Ala' and the reagents were scaled accordingly. For a typical synthesis, 2- chlorotrityl resin ( mesh; 0.6 mmol substitution/g; 25 µmol) was added to a dry RV. Two successive 15 min incubations with 5 ml dimethylformamide (DMF) and magnetic stirring were used to swell the resin. After swelling, DMF was removed with vacuum suction. The first amino acid in DMF (5 equiv; 83 mm, 1.5 ml) and DIPEA (10 equiv; 44 µl) was added to the RV and the mixture was allowed to react for 30 min with magnetic stirring. Spent solution was removed with vacuum suction and the resin beads were washed thoroughly with DMF. Excess DMF was removed with vacuum suction and the resin beads were deprotected by treatment with 20% piperidine in DMF (5 ml) for 20 min with magnetic stirring. The deprotection solution was drained from the RV and the beads were rinsed extensively with DMF. Subsequent amino acid couplings and deprotections proceeded as described above, with the exception that the remaining Fmoc-protected amino acids were activated with HBTU (5 equiv) prior to addition to each reaction. Thioamide-containing amino acids were introduced through pre-activated protected-benzotriazolides so HBTU was not added for these couplings. The final N-terminal S3

4 Fmoc group was removed before the peptides were cleaved from the resin. After the beads were washed extensively with DMF and dried with CH 2 Cl 2, peptides were cleaved by successive 60 min and 30 min incubations on a rotisserie with 2.5 ml of a fresh cleavage cocktail of trifluoroacetic acid (TFA), water, and triisopropylsilane (TIPS) (10:9:1 v/v). For AKGL'AAFAµ, a cleavage cocktail of 5% TFA in CH 2 Cl 2 with 2.5% TIPS was used. After each treatment, the resulting solution was expelled from the RV with nitrogen, reduced to a volume of less than 1 ml by rotary evaporation, and diluted with 6 ml of CH 3 CN/H 2 O (2:1 v/v). The peptides were purified to homogeneity by reverse-phase HPLC on a Vydac 218TP C18 semi-prep column (Grace/Vydac; Deerfield, IL, USA) using the following linear solvent gradients: For F'aa and the AAFAµ peptides, the gradient was isocratic at 98% aqueous phase for 6 min and then ranged from 98% to 60% aqueous phase over 19 min, then to 0% aqueous phase over 5 min, then returning to 98% aqueous phase during a 10 min wash out period. F'aa eluted around 18 min and the AAFAµ peptides eluted around 23 min. For the LPFAERµ peptides, the gradient was isocratic at 98% aqueous phase for 6 min and then ranged from 98% to 70% aqueous phase over 6 min, then to 50% aqueous phase over 20 min, then to 0% aqueous phase over 5 min, then returning to 98% aqueous phase during a 10 min wash out period. The LPLFAERµ peptides eluted around 17 min with this method. For the LLKAAµ peptides, the gradient was isocratic at 98% aqueous phase for 6 min and then ranged from 98% to 70% aqueous phase over 14 min, then to 60% aqueous phase over 10 min, then to 0% aqueous phase over 5 min, then returning to 98% aqueous phase during a 10 min wash out period. The LLKAAµ peptides eluted around 22 min with this method. For the AKGLAAFAµ peptides, the gradient was isocratic at 98% aqueous phase for 6 min and then ranged from 98% to 67% aqueous phase over 4 min, then to 63% aqueous phase over 15 min, then to 0% aqueous phase S4

5 over 5 min, then returning to 98% aqueous phase during a 5 min wash out period. The AKGLAAFAµ peptides eluted around 15 min with this method. MALDI-MS was used to confirm peptide identities. (Table S1) Purified peptides were dried in a vacuum centrifuge (Savant/Thermo Scientific; Rockford, IL, USA). Table S1. Observed and Calculated Peptide Masses. Peptide Calculated [M+H] + Observed [M+H] + Calculated [M+Na] + Observed [M+Na] + AAFAµ A'AFAµ LLKAAµ L'LKAAµ AKGLAAFAµ AKGL'AAFAµ LPLFAERµ L'PLFAERµ F'aa Biotin~F'aa Biotin~µ S5

6 Biotinylated Substrate Synthesis. Biotin~F'aa (compound S3) was prepared by combining approximately 0.5 µmol F'aa peptide (compound S1) with an approximately stoichiometric amount of EZ-Link Sulfo-NHS-Biotin (compound S2) in 250 µl sodium phosphate buffer (20 mm, ph 7.20). The mixture was allowed to react at room temperature for 2 h and was purified to homogeneity by HPLC using the same gradient described above for the F'aa peptide. With this method, the peptide conjugate eluted at approximately 27 min. MALDI- MS was used to confirm identity. (Table S1) Biotin~µ (compound S6) was prepared analogously. First, 82.2 mg Fmoc-β-(7- methoxycoumarin-4-yl)-ala-oh (S4) was dissolved in a solution of diethylamine in CH 2 Cl 2 (1:1 v/v) and allowed to stir at room temperature for 3 h. The resulting mixture was purified to homogeneity by HPLC with a Vydac 218TP C18 semi-prep column and a linear solvent gradient that was isocratic for 6 min at 98 % aqueous phase and then ramped from 98 % to 80 % aqueous phase over 4 min, then to 65 % aqueous phase over 15 min, then to 0 % aqueous phase over 5 min, where it was held for 5 min before returning to 98% aqueous phase during a 5 min wash out period. With this method, compound S5 eluted at 13 min. MALDI-MS was used to confirm peak identities (m/z calcd for C 13 H 14 NO + 5 [M+H] , found ). Fractions containing the desired product were combined and lyophilized to dryness. The solid was reconstituted in 2 ml 20 mm sodium phosphate buffer ph 7.20 and combined with an approximately stoichiometric amount of compound S2. The mixture was allowed to react at room temperature for 2 h and was then purified to homogeneity by HPLC using the following linear solvent gradient: The gradient was isocratic at 98% aqueous phase for 6 min and then ranged from 98% to 50% aqueous phase over 14 min, then to 0% aqueous phase over 5 min, then returning to 98% aqueous phase during a 10 min wash out period. Biotin~µ eluted around 20 min. MALDI-MS S6

7 was used to confirm peak identities. (Table S1) Fractions containing the desired product were combined and lyophilized. Scheme S1. Synthesis of Biotin~F'aa and Biotin~µ. S7

8 Figure S1. Steady State Protease Assays. Three independent trials for each enzyme are shown as open circles, thin lines, and thick lines. Fluorescence traces of the thiopeptide in the presence (red trace) and absence (orange trace) of protease are shown with those for the corresponding oxopeptide also in the presence (green trace) and absence (blue trace) of protease. Conditions are described in text. S8

9 Papain HPLC Assay. Papain reactions (200 µl total volume; 26.7 µm LLKAAµ or L'LKAAµ in 2.0 mm EDTA, 5.00 mm L-cysteine, 300 mm NaCl, ph 6.2 at 25 C in the presence and absence of 3.75 µg/ml papain) and control samples (200 µl total volume; 2.0 mm EDTA, 5.00 mm L-cysteine, 300 mm NaCl, ph 6.2 at 25 C with and without 3.75 µg/ml papain) were quenched at 10 and 100 min by addition of acetone (800 µl) to precipitate the protease. The reactions were cooled at -20 C for 1 h and then centrifuged at 13,200 rpm at 4 C for 20 min. The supernatant was transferred to a clean microcentrifuge tube, which was left uncapped overnight in a fume hood to allow the acetone to evaporate. The next day, the remaining solutions were dried in a vacuum centrifuge. Each sample was brought up in 1 ml H 2 O and analyzed by HPLC with a Phenomenex Luna C8(2) analytical column (Torrance, CA, USA) using a linear solvent gradient that was isocratic for 6 min at 98 % aqueous phase and then ramped from 98 % to 80 % aqueous phase over 4 min, then to 69 % aqueous phase over 11 min, then to 0 % aqueous phase over 4 min, where it was held for 5 min before returning to 98% aqueous phase during a 5 min wash out period. Absorbance was monitored at 277 nm and 325 nm. MALDI-MS was used to confirm the identity of peaks. The control samples were identical, and the average signal from the two controls as a function of time was subtracted from all other data sets to generate the chromatograms shown. (Fig. S2) S9

10 Figure S2. HPLC Analysis of Papain Reactions. Reactions in the presence (+) and absence (-) of papain monitored at 277 nm (blue trace) and 325 nm (red trace) at 10 min (left) and 100 min (right). Solvent gradient given in text. Peaks corresponding to the intact peptides LLKAAµ and L'LKAAµ are indicated by squares and circles, respectively. Peaks corresponding to the hydrolyzed fragments AAµ and L'LK are labeled with stars and triangles, respectively. Masses determined by MALDI-MS are listed for each peak and match those calculated for the expected products: For LLKAAµ [M+H] + calcd , found ; for L'LKAAµ [M+H] + calcd , found ; for AAµ [M+H] + calcd , found ; for L'LK [M+H] + calcd , found S10

11 Heat-Deactivated Enzyme Assay. A sample of papain was heat-deactivated at 95 C for 10 min. The steady state papain assay described above was repeated at 25 C with active and inactive enzyme. No change in fluorescence was observed for the thiopeptide L'LKAAµ in the presence of the denatured enzyme. The results obtained with the active enzyme were analogous to those previously observed. (Fig. S3) Figure S3. Papain Assay with Deactivated Enzyme. Fluorescence of 8.3 µm LLKAAµ or L'LKAAµ in 2.0 mm EDTA, 5.00 mm L-cysteine, 300 mm NaCl, ph 6.2 at 25 C in the presence and absence of 2.5 µg/ml papain or in the presence of heat-deactivated papain prepared by heating the enzyme in buffer at 95 C for 10 min. Traces are colored to match to those in Figure 2: The fluorescence of the thiopeptide in the presence (red trace) and absence (orange trace) of active protease is shown with the corresponding oxopeptide also in the presence (green trace) and absence (blue trace) of active protease. The gray trace shows the fluorescence of the thiopeptide in the presence of inactive enzyme. The excitation wavelength was 325 nm and the emission wavelength was 390 nm. S11

12 Substrate Specificity Assay. Papain preferentially cleaves sequences with the three residue motif: [hydrophobic amino acid]-[lys or Arg]-[not valine]. 1 To explore substrate specificity, we repeated the papain assay with the AAFAµ and A'AFAµ peptides, which we did not expect to be cleaved as efficiently as the LLKAAµ substrate. We saw no evidence of proteolysis for the AAFAµ peptides. (Fig. S4) Figure S4. Papain Substrate Specificity. Fluorescence of 8.3 µm AAFAµ or A'AFAµ in 2.0 mm EDTA, 5.00 mm L-cysteine, 300 mm NaCl, ph 6.2 at 25 C in the presence and absence of 2.5 µg/ml papain. Traces are colored to match to those in Figure 2: The fluorescence of the thiopeptide in the presence (red trace) and absence (orange trace) of papain is shown with the corresponding oxopeptide also in the presence (green trace) and absence (blue trace) of papain. The excitation wavelength was 325 nm and the emission wavelength was 390 nm. S12

13 Stopped-Flow Kinetics Data Analysis. The concentration of cleaved thioamide peptide as a function of time was determined from fluorescence measurements. In separate experiments, we measured the fluorescence of equimolar solutions of L'LKAAµ and LLKAAµ, as well as mixtures containing both peptides at a fixed total concentration. We observed a linear relationship between the mole fraction (χ) of LLKAAµ and fluorescence intensity. (Fig. S5) Figure S5. Fluorescence of LLKAAµ. Normalized fluorescence at 390 nm of mixtures of LLKAAµ and L'LKAAµ (λ ex = 325 nm). Error bars are standard error. The data were fit to a linear equation of the form: F = χ (S1) where F was the fluorescence intensity at 390 nm and χ was the mole fraction of LLKAAµ. Using this equation, we determined the fluorescence of L'LKAAµ to be % of LLKAAµ. Since the cleaved thioamide peptide fluoresces identically to the intact oxoamide peptide, this ratio could be used to calculate the mole fraction of cleaved thiopeptide (χ thio ) in each reaction with equation S2. S13

14 χ thio = F F0 (S2) Here, F is the fluorescence of the reaction at each time point t and F 0 is the fluorescence of the intact thiopeptide. F 0 was determined by extrapolating the y-intercept from linear regressions of the first 2 s of measured fluorescence for each reaction. The concentration of cleaved peptide was calculated by multiplying the initial substrate concentration by χ thio. Plots of product concentration versus time are shown. (Fig. S6) Papain catalysis follows the mechanistic scheme shown in Figure 2. If we assume that the initial formation of enzyme-substrate complex is a rapid equilibrium characterized by the equilibrium constant K S (equation S3), then the kinetics of the system can be described by equation S4, where the instantaneous concentration of substrate [S] at time t has been taken to be approximately equal to the initial substrate concentration [S] 0 and the total enzyme concentration is [E] 0. 2 [E][S] [ ES] k k 1 K S = = (S3) 1 k 2 ( ) + = k k2 + k3 [S] 0 + k2ks t cat[e] [S] k k KS + [S] [ P] 0 t [E] e + K 0 2 m K 1 0 (S4) [S] 0,app + m,app 1 [S] 0 2 The apparent Michaelis-Menten constant is defined with equation S5 and k cat is given by equation S6. K k K m + S 3,app = (S5) k2 k3 S14

15 k k k cat k + k 2 3 = (S6) 2 3 The rapid mixing kinetics data were globally fit to equation S4 using the Solver data analysis package in Excel while treating the total active enzyme concentration [E] 0, the equilibrium constant K S, and the rate constants k 2 and k 3 as adjustable parameters. Using this method, we found [E] 0 = 1.07 µm, K S = 921 µm, k 2 = 0.92 s -1, k 3 = s -1, k cat = s -1, and K M,app = 53.9 µm. The Michaelis-Menten plot in Figure 3 was generated by calculating initial rates according to equation S7 and fitting the data with KaleidaGraph (Synergy Software; Reading, PA, USA). The fit in Kaleidagraph returned nearly identical values to those obtained from global fitting of the primary data, k cat = ± s -1 and K M,app = 50.2 ± 1.1 µm. v 0 kcat[e] 0[S] = [S] + K 0 0 m,app (S7) S15

16 Figure S6. Global Fit to Papain Rapid-Mixing Data. Fit of equation S4 to primary data as described in text. Initial substrate concentrations [S] 0 are listed for each data set. S16

17 Figure S7. Low-grade Trypsin. Fluorescence of 8.3 µm AKGLAAFAµ or AKGL'AAFAµ in 67 mm sodium phosphate buffer, ph 7.6, at 30 C in the presence and absence of 25 µg/ml technical grade trypsin. Traces are colored to match to those in Figure 2: The fluorescence of the thiopeptide in the presence (red trace) and absence (orange trace) of papain is shown with the corresponding oxopeptide also in the presence (green trace) and absence (blue trace) of technical grade trypsin. The excitation wavelength was 325 nm and the emission wavelength was 390 nm. S17

18 Figure S8. HPLC Analysis of Multi-Site Substrate Assay. Absorbance at 277 nm (blue traces) and 325 nm (red traces) of the supernatant from trypsin (left) and chymotrypsin (right) experiments with AKGLAAFAµ and AKGL'AAFAµ peptides as indicated and described in the text. Peaks corresponding to AKGLAAFAµ and AKGL'AAFAµ are labelled with squares and circles, respectively. Peaks corresponding to the peptide fragments GLAAFAµ, GL'AAFAµ, Aµ, and AKGL'AAF are labelled with stars, triangles, diamonds, and double daggers, respectively. Peaks were identified with MALDI-MS: for AKGLAAFAµ [M+H] + calcd found ; for AKGL'AAFAµ [M+H] + calcd found ; for GLAAFAµ [M+H] + calcd found ; for GL'AAFAµ [M+H] + calcd found ; for Aµ [M+H] + calcd found ; and for AKGL'AAF [M+H] + calcd found S18

19 Thiopeptide Stability. A 100 μl sample of crude A'AFAμ in CH 3 CN/H 2 O (2:1 v/v) was diluted with 900 μl H 2 O and analyzed by HPLC immediately after cleavage from the resin beads using the solvent gradient described above. A second sample of the crude peptide was stored in the same CH 3 CN/H 2 O solution at 4 C for 454 days, after which time it was brought up to 1 ml with H 2 O and purified using the same HPLC method. (Fig. S9) No differences were observed in the chromatograms between the two samples, and the major peak was identified as A'AFAμ with MALDI-MS in both cases. Figure S9. Stability of crude A'AFAµ in CH 3 CN/H 2 O over 454 days at 4 C. Absorbance monitored at 277 nm. S19

20 Thiopeptide Stability in Serum and PBS. Samples of the thioamide-containing peptide F'aa conjugated to a cleavable biotin linker (compound S3) and the internal standard Biotin~µ (compound S6) were incubated in mouse serum or PBS buffer (phosphate buffered saline, 10 mm Na 2 HPO 4, 150 mm NaCl, ph 7.00; ph adjusted with HCl) for up to 24 h at 37 C. For these experiments, approximately equimolar amounts of the peptide conjugate and internal standard were dissolved in H 2 O (Biotin~F'aa: 70.5 µm, Biotin~µ: 94.3 µm). Aliquots (70 µl) of this solution were added to 280 µl samples of mouse serum or PBS. Samples were kept in a 37 C water bath for 1 h, 4 h, or 24 h periods. Separate samples, brought up only in water, were analyzed immediately (0 h). After the appropriate incubation period, the samples were purified on an immobilized neutravadin resin. The resin was prepared by loading 400 µl of resin slurry into a fritted column and washing it with 10 column volumes (CVs) of PBS. The sample was then added to the column and allowed to incubate at room temperature for 10 min. The column was washed with 10 CVs PBS. A 1 ml portion of freshly prepared 50 mm DTT was then added to the column and allowed to incubate for 30 min at room temperature on a rotisserie. The flow through was collected and analyzed by HPLC using the gradient described above for LLKAAµ with a Vydac 218TP C18 analytical column. (Fig. S10) Fractions corresponding to the observed peaks were dried in a vacuum centrifuge and then analyzed by MALDI-MS. The peaks corresponded to the expected mass of the cleaved coumarin standard (~µ, compound S7; m/z calcd for C 16 H 18 NO 6 S + [M+H] , found ; for C 16 H 17 NNaO 6 S + [M+Na] , + found ) or the cleaved peptide (~F'aa, compound S8; m/z calcd for C 18 H 25 N 3 NaO 4 S 2 [M+Na] , found ; for C 18 H 25 KN 3 O 4 S + 2 [M+K] , found ). Three independent trials were conducted for each reaction condition. Peak areas corresponding to ~F'aa and ~µ were calculated using the integrated peak intensity at 270 nm or S20

21 325 nm, respectively. To account for the potential loss of material during the workup and purification process, the ~F'aa signal was normalized to that of ~µ in each trial. We arbitrarily assigned the average ratio of the integrated areas of these peaks to be equal to 100 for the all water (0 h) trials. The integrated peak area ratios from all other trials were scaled accordingly and then averaged to determine the mean and standard error. (Fig. S10) Using this method, we observed no loss of Biotin~F aa in PBS after 1 h, and only a 5 % loss after 24 h. In mouse serum, a 15 % loss was observed after 1 h, and a 20 % loss was observed after 4 h. S21

22 Figure S10. Thiopeptide stability. (a) Amount of ~F'aa relative to the amount of ~µ recovered after incubating a mixture of biotin~f'aa (S3) and biotin~µ (S6) in various conditions. The results are normalized to the 0 h data set. (b) Chemical structures of compounds S7 (~µ) and S8 (~F'aa) and calculated masses. (c) Representative HPLC traces of each trial. Absorbance at 325 nm (red trace) and 277 nm (blue trace) is shown. Peaks corresponding to ~µ and ~F'aa are indicated with squares and circles, respectively. Peak identities were confirmed by MALDI-MS (representative masses shown). The slight difference in retention time is the result of a change in the HPLC plumbing between PBS and serum analyses. S22

23 Purified Calpain Assays. The enzyme concentration for calpain 1 was 25 nm. Calpain buffer contained 10 mm dithioreitol (DTT), 5 mm KH 2 PO 4, 2 mm EGTA, and 0.015% Brij-35, ph ,4 The LPLFAERµ and L'PLFAERµ substrates were present at 8 µm. Calpain was activated by the addition of CaCl 2 via a multichannel pipette to a final concentration of 10 mm. The negative control was buffer addition instead of calcium addition. All assays were done at a total well volume of 100 µl in 96-well black flat bottom plate. Fluorescence intensity was read in a Tecan plate reader over 30 min. The excitation wavelength was 330 nm and the emission wavelength was 390 nm. A fluorescence increase was observed only in the case of the Ca 2+ - activated protease with thiopeptide. (Fig. S11) Figure S11. Purified Calpain Assay. Normalized fluorescence of 8 µm LPLFAERµ or L'PLFAERµ in buffer containing 25 nm calpain 1, 10 mm DTT, 5 mm KH 2 PO 4, 2 mm EGTA, and 0.015% Brij-35, ph 7.5, in the presence and absence of 10 mm CaCl 2. The fluorescence of the thiopeptide in the presence (red trace) and absence (orange trace) of calcium is shown with the corresponding oxopeptide also in the presence (green trace) and absence (blue trace) of calcium. The excitation wavelength was 330 nm and the emission wavelength was 390 nm. S23

24 Sensitivity Calculations. Signal/noise ratios for steady state (i.e., not rapid mixing) experiments were calculated by dividing the average value of fluorescence intensity counts for a thiopeptide trial in the absence of protease by the standard deviation of all the data points in that trial. Signal/background ratios were determined as the observed fluorescence signal relative to the dark counts for a comparable buffer solution. These values vary between spectrometers, where signal/background ratios were about 150 for experiments on the Cary Eclipse fluorometer and about 2000 for experiments on the Tecan M1000 plate reader. For experiments in cell lysates, signal/noise ratios were calculated by dividing the average value of the normalized fluorescence intensity counts for the A'AFAμ trial after 10 min (i.e., after proteolysis was complete) by the standard deviation of all the data points in that time period. S24

25 References (1) Garcia-Echeverria, C.; Rich, D. H. FEBS Lett. 1992, 297, (2) Bender, M. L.; Kezdy, F. J.; Wedler, F. C. J. Chem. Ed. 1967, 44, 84. (3) Kelly, J. C.; Cuerrier, D.; Graham, L. A.; Campbell, R. L.; Davies, P. L. Biochim. Biophys. Acta 2009, 1794, (4) Gil-Parrado, S.; Assfalg-Machleidt, I.; Fiorino, F.; Deluca, D.; Pfeiler, D.; Schaschke, N.; Moroder, L.; Machleidt, W. Biol. Chem. 2003, 384, S25