Supporting Information 3,4-Dihydroxyphenylalanine Peptides as. Nonperturbative Quantum Dot Sensors of

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1 Supporting Information 3,4-Dihydroxyphenylalanine Peptides as Nonperturbative Quantum Dot Sensors of Aminopeptidase Valle Palomo 1, Sebastián A. Díaz 2, Michael H. Stewart 3, Kimihiro Susumu 3, Igor L. Medintz 2, Philip E. Dawson*,1. 1 Department of Chemistry, The Scripps Research Institute, La Jolla, California, 92037, United States 2 Center for Bio/Molecular Science and Engineering, Code 6900, and 3 Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory, Washington, D.C., 20375, United States

2 Table of Contents. Fig. S1: QD photoluminescence is quenched by self-assembled DOPA containing peptides..3 Fig. S2: Peptide fragmentation and Mass observed after chymotrypsin digestion of RAXARK.. 4 Fig. S3: Peptide fragmentation and Mass observed after chymotrypsin digestion of peptide 1 4 Fig. S4: Reconstructed mass of the fragments encountered after digestion of peptide 7 with LAP 5 Fig. S5: Photoluminescence is restored after chymotrypsin treatment Fig. S6. Photoluminescence is restored after LAP treatment....6 Methodology for kinetic calculations. 7

3 Figure S1. QD photoluminescence is quenched by self-assembled DOPA containing peptides. Photoluminescence spectra collected from 523-nm-emitting DHLA-PEG-QDs self-assembled with an increasing ratio of 3 added to 2 mm Na 2 B 4 O 7 buffer at ph 4.5 (A), ph 7.0 (B) and 9.5 (C). Spectra were collected on an AVIV spectrofluorometer with 350 nm excitation. (D) Stern-Volmer plots of an increasing ratio of DOPA peptide per QD versus I 0 /I performed at ph 4.5, 7.0 and 9.5 (data from A,B and C). Values in parenthesis are slopes derived for each data set reflecting the higher quenching ratio for increasing ph values.

4 m/z Figure S2. Peptide fragmentation and mass observed after chymotrypsin digestion of RAXARK m/z Figure S3. Peptide fragmentation and mass observed after chymotrypsin digestion of peptide 1.

5 Figure S4. Reconstructed mass of the fragments encountered after digestion of peptide 7 with LAP. Fragments correspond to peptide 7 (2369) peptide 7 minus Arg (2213); peptide 7 minus Arg-Ala (2142); peptide 7 minus Arg-Ala-DOPA (1963) and peptide 7 minus Arg-Ala-DOPA-Ala-Arg-Lys-Ala (1537). The different colors represent the different peaks encountered in the HPLC trace. Figure S5. Photoluminescence is restored after chymotrypsin treatment. Normalized photoluminescence spectra of SQQDs at different DHLA-CL4 and DHLA-PEG- QD/peptide ratio before and after peptidase treatment. (A) DHLA-CL4 coated QDs

6 loaded with peptide 6 at 0, 10, 18 and 36 peptides per QD ratio. (B) DHLA-CL4 coated QDS loaded peptide 6 at 0, 10, 18 and 36 peptides per QD ratio after 1 h of chymotrypsin incubation at 1.6 μm at 37 ºC. (C) DHLA-PEG coated QDs loaded with peptide 1 at 0, 10, 18 and 36 peptides per QD ratio. (D) DHLA-PEG coated QDs loaded with peptide 1 at 0, 10, 18 and 36 peptides per QD ratio after 1 h of chymotrypsin incubation at 1.6 μm at 37 ºC. Figure S6. Photoluminescence is restored after LAP treatment. Normalized photoluminescence spectra of SQQDs at different DHLA-PEG-QD/peptide ratio before and after peptidase treatment. (A), (C), (E), (G) QDs loaded with specified peptide at 0,

7 18 and 36 peptides per QD ratio. (B), (D), (F), (H) QDs loaded specified peptide at 0, 18 and 36 peptides per QD ratio after 1 h of LAP incubation at 0.5 μm at 37 ºC. (A) and (B) peptide 2; (C) and (D) peptide 7; (E) and (F) peptide 3. Methodology for kinetic calculations The raw progress curves were normalized to a control run of zero enzyme to correct for instrumental drift and then transformed into peptide/qd ratios utilizing the calibrations values. Having a known QD concentration the curves were converted into substrate consumption as a function of time. By multiplying the time by the enzyme concentration of each well we obtained the enzyme time progress curves. The data points were combined and analyzed by using the integrated solution to the Michaelis- Menten (MM) equations: k cat Et = p + K m ln ( a 0 ) (Eq. S1) a 0 p Where k cat and K m are the catalytic constant and Michaelis constant respectively. The reaction product is represented as p and the initial substrate as a 0. E is the enzyme concentration and t represents time, allowing us to represent the data as enzyme time Et = E t. During the integration of the progress curves, p is dependent on the point of the reaction course, therefore a non-linear fit with a numerical solution is required; these were realized by an in-house developed Wolfram Mathematica 10.0 notebook (Wolfram Inc.) using: p = K m ProductLog ( a 0 K m e ( a0 K cat Et Km ) ) (Eq. S2) Where the ProductLog represents the Lambert W-function. The fits were capable of obtaining the k cat /K m ratio, known as the specificity constant, of 50 ± 15 mm -1 s -1 for LAP and the DOPA peptide. Similar to what is observed in classical fixed enzyme experiments to determine the individual k cat and K m parameters a larger range of concentrations must be tested. This higher concentration range is inaccessible due to the limited solubility of the colloidal nanoparticles, limiting us to the specificity constant value.