DOI: 10.5061/dryad.fttdz092d
title: Data from: Reversible and size-controlled assembly of reflectin proteins using a charged azobenzene photoswitch

description: Disordered proteins often undergo a stimuli-responsive, disorder-to-order
 transition which facilitates dynamic processes that modulate the
 physiological activities and material properties of cells, such as
 strength, chemical composition, and reflectance. It remains challenging to
 gain rapid and spatiotemporal control over such disorder-to-order
 transitions, which limits the incorporation of these proteins into novel
 materials. The reflectin protein is a cationic, disordered protein whose
 assembly is responsible for dynamic color camouflage in cephalopods.
 Stimuli-responsive control of reflectin’s assembly would enable the design
 of biophotonic materials with tunable color. Herein, a novel, multivalent
 azobenzene photoswitch is shown to be an effective and non-invasive
 strategy for co-assembling with reflectin molecules and reversibly
 controlling assembly size. Photoisomerization between the trans and cis (E
 and Z) photoisomers promotes or reduces Coulombic interactions,
 respectively, with reflectin proteins to repeatedly cycle the sizes of the
 photoswitch-reflectin assemblies between 70 nm and 40 nm. The protein
 assemblies formed with the trans and cis isomers show differences in
 interaction stoichiometry and secondary structure, which indi-cate that
 photoisomerization modulates the photoswitch-protein interactions to
 change assembly size. Our results high-light the utility of
 photoswitchable interactions to control reflectin assembly and provide a
 tunable synthetic platform that can be adapted to the structure, assembly,
 and function of other disordered proteins.
description: Synthetic procedures and characterization, reflectin protein
 purification and methods for sample preparation, additional
 characterization of protein-photoswitch complexes.
description: # Reversible and Size-Controlled Assembly of Reflectin Proteins Using a
 Charged Azobenzene Photoswitch Supporting Data
 [https://doi.org/10.5061/dryad.fttdz092d](https://doi.org/10.5061/dryad.fttdz092d) ## Description of the data and file structure Data from peer-reviewed article: Title: Reversible and Size-Controlled Assembly of Reflectin Proteins Using a Charged Azobenzene Switch Article: Chemical Science Authors: Cassidy M. Tobin, Reid Gordon, Seren K. Tochikura, Bradley F. Chmelka, Daniel E. Morse, and Javier Read de Alaniz Corresponding author: Javier Read de Alaniz, javier@chem.ucsb.edu \------------------------------------------------------------------------------------------------------------------------------------- File List A) Figure 2A B) Figure 2D C) Figure 3B D) Figure 3CandS13 E) Figure 3D F) Figure 4B G) Figure 4C H) Figure4C_PrecipitationExp_TransAzoEDTA I) Figure4C_PrecipitationExp_CisAzoEDTA J) Figure 4D K) Figure 5B L) Figure 5C M) Figure 5D N) Figure 5E O) Figure 5F P) Figure S1 Q) Figure S2B R) Figure S3 S) Figure S4A T) Figure S4B U) Figure S5 V) Figure S6A W) Figure S6B X) Figure S7 Y) Figure S8 Z) Figure S9B AA) Figure S10 AB) Figure S11 AC) Figure S12A AD) Figure S12B AE) Figure S14 AF) Figure S15A AG) Figure S15B AH) Figure S17 AI) Figure S18 AJ) Figure S19 AK) Figure S20A AL) Figure S20B AM) Figure S20C AN) Figure S20D AO) Figure S21A AP) Figure S21B AQ) Figure S22 AR) Figure S23 AS) Figure S24 AT) Figure S25 AU) Figure S26 AV) Figure S27 AW) Figure S28 AX) Figure S29 AY) Figure S30 Note: Figures 3D, 4C-D, 5B, S2B, S9B, S11, S19, S22, S25 all contain "n/a" at the bottom of several columns. This is due to different numbers of rows between different datasets given in each text file. \------------------------------------------------------------------------------------------------------------------------------------- Figure 2A: Hydrodynamic diameter of reflectin assemblies with various multivalent acids File: Figure2A.csv \* Column A: Name of multivalent acid used in assembly \* Column B: Mean hydrodynamic diameter (nm) of reflectin-acid assemblies \* Column C: Standard deviation of mean diameter (nm) \------------------------------------------------------------------------------------------------------------------------------------- Figure 2D: Normalized absorbance spectra of 320 uM azoEDTA photoswitch before and after irradiation with 365 nm and 470 nm light File: Figure2D.csv \* Column A: Wavelength (nm) \* Column B: Absorbance of azoEDTA prior to irradiation (stored in dark for 3 days) \* Column C: Absorbance of azoEDTA after 20 min of irradiation with 365 nm light \* Column D: Absorbance of azoEDTA after 8 min of irradiation with 470 nm light Data in figure was normalized by the wavelength of maximum absorption (327 nm) in Column B dataset \------------------------------------------------------------------------------------------------------------------------------------- Figure 3B: Turbidity of azoEDTA-reflectin samples as a function of azoEDTA concentration File: Figure3B.csv \* Column A: AzoEDTA Concentration (uM) \* Column B: Turbidity (%) of solution Turbidity was calculated from the absorbance value at 400 nm, a wavelength where azoEDTA has a negligible absorbance. The absorbance spectra used for this plot can be found in the Figure 4C dataset. \------------------------------------------------------------------------------------------------------------------------------------- Figure 3C, S13: Mean hydrodynamic diameter of azoEDTA-reflectin assemblies as a function of azoEDTA concentration File: Figure3CandS13.csv Data in Figures 3C and S13 are identical except that S13 includes concentrations of azoEDTA &gt; 500 uM. The data are shown here as one dataset. \* Column A: Concentration of azoEDTA (uM) \* Column B: Mean hydrodynamic diameter of protein assemblies (nm) \* Column C: Standard deviation of the mean diameter (nm) \------------------------------------------------------------------------------------------------------------------------------------- Figure 3D: Ellipticity of azoEDTA-reflectin assemblies at different azoEDTA concentrations, taken with circular dichroism spectroscopy File: Figure3D.csv \* Column A: Wavelength (nm), for Column B data \* Column B: Ellipticity (mdeg) of 8 uM, monomeric reflectin at pH 4.50 \* Column C: Wavelength (nm), for Columns D-G data \* Column D: Ellipticity (mdeg) for azoEDTA-reflectin assembly prepared with 16 uM azoEDTA and 8 uM reflectin \* Column E: Ellipticity (mdeg) for azoEDTA-reflectin assembly prepared with 184 uM azoEDTA and 8 uM reflectin \* Column F: Ellipticity (mdeg) for azoEDTA-reflectin assembly prepared with 320 uM azoEDTA and 8 uM reflectin \* Column G: Ellipticity (mdeg) for azoEDTA-reflectin assembly prepared with 480 uM azoEDTA and 8 uM reflectin \------------------------------------------------------------------------------------------------------------------------------------- Figure 4B: Logarithmic plot of mean hydrodynamic diameter of azoEDTA-reflectin assemblies with 240 uM azoEDTA File: Figure4B.csv \* Column A: Hydrodynamic Diameter (nm) \* Column B: Volume (%) distribution of trans-azoEDTA and reflectin assemblies \* Column C: Volume (%) distribution of cis-azoEDTA and reflectin assemblies \------------------------------------------------------------------------------------------------------------------------------------- Figure 4C: Interaction stoichiometry - mole of azoEDTA per mole of protein in the azoEDTA-reflectin assembled (i.e, dense) phase File: Figure4C.csv AzoEDTA-reflectin assemblies prepared with trans-azoEDTA: \* Column A: Concentration of trans-azoEDTA (uM) in azoEDTA-reflectin assemblies \* Column B: Mean interaction stoichiometry of trans-azoEDTA and reflectin, averaged from 3 replicates \* Column C: Standard deviation of Column B AzoEDTA-reflectin assemblies prepared with cis-azoEDTA: \* Column D: Concentration of cis-azoEDTA (uM) in azoEDTA-reflectin assemblies \* Column E: Mean interaction stoichiometry of cis-azoEDTA and reflectin, averaged from 3 replicates \* Column F: Standard deviation of Column E The equations used to calculate the interaction stoichiometry can be found in the Supplemental Information. The absorbance spectra collected to calculate the data shown in Figures 3B, 4C, 4D, and S19 are given below For assemblies prepared with the trans-azoEDTA photoisomer: File: Figure4C_PrecipitationExp_TransAzoEDTA.csv \* Column A: Wavelength (nm) \* Column B: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 80 uM trans-azoEDTA, replicate 1 \* Column C: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 80 uM trans-azoEDTA, replicate 2 \* Column D: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 80 uM trans-azoEDTA, replicate 3 \* Column E: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 160 uM trans-azoEDTA, replicate 1 \* Column F: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 160 uM trans-azoEDTA, replicate 2 \* Column G: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 160 uM trans-azoEDTA, replicate 3 \* Column H: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 240 uM trans-azoEDTA, replicate 1 \* Column I: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 240 uM trans-azoEDTA, replicate 2 \* Column J: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 240 uM trans-azoEDTA, replicate 3 \* Column K: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 320 uM trans-azoEDTA, replicate 1 \* Column L: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 320 uM trans-azoEDTA, replicate 2 \* Column M: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 320 uM trans-azoEDTA, replicate 3 \* Column N: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 400 uM trans-azoEDTA, replicate 1 \* Column O: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 400 uM trans-azoEDTA, replicate 2 \* Column P: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 400 uM trans-azoEDTA, replicate 3 \* Column Q: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 480 uM trans-azoEDTA, replicate 1 \* Column R: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 480 uM trans-azoEDTA, replicate 2 \* Column S: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 480 uM trans-azoEDTA, replicate 3 For assemblies prepared with the cis-azoEDTA photoisomer: File: Figure4C_PrecipitationExp_CisAzoEDTA.csv \* Column A: Wavelength (nm) \* Column B: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 160 uM trans-azoEDTA, replicate 1 \* Column C: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 160 uM trans-azoEDTA, replicate 2 \* Column D: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 160 uM trans-azoEDTA, replicate 3 \* Column E: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 240 uM trans-azoEDTA, replicate 1 \* Column F: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 240 uM trans-azoEDTA, replicate 2 \* Column G: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 240 uM trans-azoEDTA, replicate 3 \* Column H: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 320 uM trans-azoEDTA, replicate 1 \* Column I: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 320 uM trans-azoEDTA, replicate 2 \* Column J: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 320 uM trans-azoEDTA, replicate 3 \* Column K: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 400 uM trans-azoEDTA, replicate 1 \* Column L: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 400 uM trans-azoEDTA, replicate 2 \* Column M: Absorption spectrum of azoEDTA-reflectin assemblies prepared with 400 uM trans-azoEDTA, replicate 3 \------------------------------------------------------------------------------------------------------------------------------------- Figure 4D: Percent of soluble (non-interacting) reflectin in the supernatant following centrifugation File: Figure4D.csv AzoEDTA-reflectin assemblies prepared with trans-azoEDTA: \* Column A: Concentration of trans-azoEDTA (uM) in azoEDTA-reflectin assemblies \* Column B: Mean fraction of reflectin remaining in supernatant, averaged from 3 replicates \* Column C: Standard deviation of Column B AzoEDTA-reflectin assemblies prepared with cis-azoEDTA: \* Column D: Concentration of cis-azoEDTA (uM) in azoEDTA-reflectin assemblies \* Column E: Mean fraction of reflectin remaining in supernatant, averaged from 3 replicates \* Column F: Standard deviation of Column E The absorbance spectra used for this plot can be found in the Figure 4C dataset. The equations used to calculate the fraction of reflectin protein in the supernatant can be found in the Supplementary Information. \------------------------------------------------------------------------------------------------------------------------------------- Figure 5B: Time-dependent absorbance of azoEDTA-reflectin complexes prepared with 8 uM reflectin and 240 uM azoEDTA. File: Figure5B.csv \* Column A: Time (min) for Column B [0 min to 44.2 min] \* Column B: Sample absorbance at 338 nm during 365 nm irradiation cycle 1 \* Column C: Time (min) for Column D [45.7 min to 72.4 min] \* Column D: Sample absorbance at 338 nm during 470 nm irradiation cycle 1 \* Column E: Time (min) for Column F [73.9 min to 118.1 min] \* Column F: Sample absorbance at 338 nm during 365 nm irradiation cycle 2 \* Column G: Time (min) for Column H [119.6 min to 146.3 min] \* Column H: Sample absorbance at 338 nm during 470 nm irradiation cycle 2 \* Column I: Time (min) for Column J [147.8 min to 192.0 min] \* Column J: Sample absorbance at 338 nm during 365 nm irradiation cycle 3 \* Column K: Time (min) for Column L [193.5 min to 220.2 min] \* Column L: Sample absorbance at 338 nm during 365 nm irradiation cycle 3 Irradiation with 365 nm and 470 nm light were cycled three times and the 6 datasets are stacked sequentially. Used a 2 mm path length quartz cuvette and 350 uL of solution. 365 nm light irradiation: \* There was no light irradiation during the first 10 data points (30 seconds) collected. \* The sample was irradiated with 365 nm light for 2,025 seconds and an absorption spectrum was collected every 3 seconds. \* After irradiation, the sample remained in the dark for 600 seconds and a spectrum was collected every 5 seconds. 470 nm light irradiation: \* There was no light irradiation during the first 10 data points (30 seconds) collected. \* The sample was irradiated with 365 nm light for 975 seconds and an absorption spectrum was collected every 3 seconds. \* After irradiation, the sample remained in the dark for 600 seconds and a spectrum was collected every 5 seconds. In Figure 5B, these periods of 365 nm, 470 nm, and no irradiation are denoted as the purple, blue, and gray shadows, respectively. The full absorbance spectra for all times provided in this dataset are available upon request. Spectra taken on home-built time-dependent UV-vis. \------------------------------------------------------------------------------------------------------------------------------------- Figure 5C: Logarithmic plot of hydrodynamic diameter versus volume intensity of azoEDTA-reflectin assemblies File: Figure5C.csv \* Column A: Hydrodynamic diameter (nm) \* Column B: Volume distribution of azoEDTA-reflectin assemblies prior to irradiation (stored in dark for 120 min) \* Column C: Volume distribution of azoEDTA-reflectin assemblies after 20 min of irradiation with 365 nm light \* Column D: Volume distribution of azoEDTA-reflectin assemblies after 8 min of irradiation with 470 nm light \------------------------------------------------------------------------------------------------------------------------------------- Figure 5D: Time versus mean hydrodynamic diameter of azoEDTA-reflectin assemblies over 2 irradiation cycles File: Figure5D.csv \* Column A: Time (min) \* Column B: Mean hydrodynamic diameter (nm) of azoEDTA reflectin assemblies \------------------------------------------------------------------------------------------------------------------------------------- Figure 5E: Secondary structure evolution of azoEDTA-reflectin assemblies following 2 cycles of irradiation File: Figure5E.csv \* Column A: Wavelength (nm) \* Column B: Ellipticity (mdeg) of azoEDTA-reflectin assemblies prior to irradiation (stored in dark for 120 min) \* Column C: Ellipticity (mdeg) of azoEDTA-reflectin assemblies after 20 min of irradiation with 365 nm light, cycle 1 \* Column D: Ellipticity (mdeg) of azoEDTA-reflectin assemblies after 8 min of irradiation with 470 nm light, cycle 1 \* Column E: Ellipticity (mdeg) of azoEDTA-reflectin assemblies after 20 min of irradiation with 365 nm light, cycle 2 \* Column F: Ellipticity (mdeg) of azoEDTA-reflectin assemblies after 8 min of irradiation with 470 nm light, cycle 2 \------------------------------------------------------------------------------------------------------------------------------------- Figure 5F: Time versus ellipticity (at 202 nm) of azoEDTA-reflectin assemblies over 2 irradiation cycles File: Figure5F.csv \* Column A: Time (min) \* Column B: Ellipticity (mdeg) of azoEDTA reflectin assemblies, averaged over 4 measurements \------------------------------------------------------------------------------------------------------------------------------------- Figure S1: Net charge of the reflectin A1 wildtype protein as a function of pH File: FigureS1.csv \* Column A: pH \* Column B: Net charge of reflectin A1 wildtype protein \------------------------------------------------------------------------------------------------------------------------------------- Figure S2B: Mean hydrodynamic diameter of reflectin assemblies formed with multianionic small molecules File: FigureS2B.csv \* Column A: Acid concentration (uM) for Columns B-E \* Column B: Mean hydrodynamic diameter of assemblies formed with pentetic acid \* Column C: Standard deviation of values from Column B \* Column D: Mean hydrodynamic diameter of assemblies formed with ethylenediaminetetracetic acid \* Column E: Standard deviation of values from Column D \* Column F: Acid concentration (uM) for Columns G-J \* Column G: Mean hydrodynamic diameter of assemblies formed with suberic acid \* Column H: Standard deviation of values from Column G \* Column I: Mean hydrodynamic diameter of assemblies formed with succinic acid \* Column J: Standard deviation of values from Column I \------------------------------------------------------------------------------------------------------------------------------------- Figure S3: Mean hydrodynamic diameter of EDTA-reflectin assemblies as a function of pH and concentration of EDTA File: FigureS3.csv \* Column A: Concentration of EDTA (uM) \* Column B: Mean hydrodynamic diameter (nm) of EDTA-reflectin assemblies at pH 4.50 \* Column C: Standard deviation of Column B \* Column D: Mean hydrodynamic diameter (nm) of EDTA-reflectin assemblies at pH 7.50 \* Column E: Standard deviation of Column D \------------------------------------------------------------------------------------------------------------------------------------- Figure S4A: Logarithmic plot of volume-based size distribution of EDTA- and NaCl-reflectin assemblies at pH 4.50 File: FigureS4A.csv \* Column A: Hydrodynamic diameter (nm) \* Column B: Volume size distribution of 20 uM of reflectin \* Column C: Volume size distribution of 20 uM reflectin with 1 mM EDTA \* Column D: Volume size distribution of 20 uM reflection with 6 mM NaCl \------------------------------------------------------------------------------------------------------------------------------------- Figure S4B: Logarithmic plot of volume-based size distribution of EDTA- and NaCl-reflectin assemblies at pH 7.50 File: FigureS4B.csv \* Column A: Hydrodynamic diameter (nm) \* Column B: Volume size distribution of 20 uM of reflectin \* Column C: Volume size distribution of 20 uM reflectin with 1 mM EDTA \* Column D: Volume size distribution of 20 uM reflection with 6 mM NaCl \------------------------------------------------------------------------------------------------------------------------------------- Figure S5: pH titration of azoEDTA with hydrochloric acid File: FigureS5.csv \* Column A: umoles of hydrochloric acid added \* Column B: pH \------------------------------------------------------------------------------------------------------------------------------------- Figure S6A: Absorption spectra of azoEDTA in a 20 mM, pH 4.50 acetate buffer, taken on a NanoDrop instrument File: Figure6A.csv \* Column A: Wavelength (nm) \* Column B: Absorbance of 60 uM azoEDTA in 20 mM, pH 4.50 acetate buffer \* Column C: Absorbance of 75 uM azoEDTA in 20 mM, pH 4.50 acetate buffer \* Column D: Absorbance of 100 uM azoEDTA in 20 mM, pH 4.50 acetate buffer \* Column E: Absorbance of 125 uM azoEDTA in 20 mM, pH 4.50 acetate buffer \* Column F: Absorbance of 307 uM azoEDTA in 20 mM, pH 4.50 acetate buffer \* Column G: Absorbance of 538 uM azoEDTA in 20 mM, pH 4.50 acetate buffer \* Column H: Absorbance of 767 uM azoEDTA in 20 mM, pH 4.50 acetate buffer \------------------------------------------------------------------------------------------------------------------------------------- Figure S6B: Absorbance versus concentration of azoEDTA spectra File: Figure6B.csv \* Column A: Concentration of azoEDTA (M) \* Column B: Absorbance at 280 nm \* Column C: Absorbance at 331 nm Figure 6C was generated with this same data \------------------------------------------------------------------------------------------------------------------------------------- Figure S7: Photostationary state of azoEDTA with 365 nm and 470 nm light irradiation via H-NMR File: FigureS7.csv \* Column A: Chemical Shift (ppm) \* Column B: Signal intensity of 3.1 mM azoEDTA in 10 vol% D2O, thermally equilibrated in the dark for 3 days \* Column C: Signal intensity of 3.1 mM azoEDTA in 10 vol% D2O, after 40 minutes of 365 nm irradiation \* Column D: Signal intensity of 3.1 mM azoEDTA in 10 vol% D2O, after 15 minutes of 470 nm irradiation \------------------------------------------------------------------------------------------------------------------------------------- Figure S8: Half-life of the cis-azoEDTA photoisomer via H-NMR File: FigureS8.csv \* Column A: Time (days) \* Column B: Fraction of cis-azoEDTA via relative integration of trans and cis amide protons on the H-NMR \* Column C: Fraction of cis-azoEDTA via relative integration of trans and cis aromatic protons on the H-NMR \------------------------------------------------------------------------------------------------------------------------------------- Figure S9B: Time-dependent absorbance of 240 uM azoEDTA. File: FigureS9B.csv \* Column A: Time (min) for Column B [0 min to 25.45 min] \* Column B: Sample absorbance at 338 nm during 365 nm irradiation cycle 1 \* Column C: Time (min) for Column D [26.95 min to 42.4 min] \* Column D: Sample absorbance at 338 nm during 470 nm irradiation cycle 1 \* Column E: Time (min) for Column F [43.9 min to 69.35 min] \* Column F: Sample absorbance at 338 nm during 365 nm irradiation cycle 2 \* Column G: Time (min) for Column H [70.85 min to 86.3 min] \* Column H: Sample absorbance at 338 nm during 470 nm irradiation cycle 2 \* Column I: Time (min) for Column J [87.8 min to 105.25 min] \* Column J: Sample absorbance at 338 nm during 365 nm irradiation cycle 3 \* Column K: Time (min) for Column L [106.75 min to 114.2 min] \* Column L: Sample absorbance at 338 nm during 365 nm irradiation cycle 3 \* Column M: Time (min) for Column F [115.7 min to 133.15 min] \* Column N: Sample absorbance at 338 nm during 365 nm irradiation cycle 4 \* Column O: Time (min) for Column H [134.65 min to 142.1 min] \* Column P: Sample absorbance at 338 nm during 470 nm irradiation cycle 4 \* Column Q: Time (min) for Column J [143.6 min to 161.05 min] \* Column R: Sample absorbance at 338 nm during 365 nm irradiation cycle 5 \* Column S: Time (min) for Column L [162.55 min to 170 min] \* Column T: Sample absorbance at 338 nm during 365 nm irradiation cycle 5 Irradiation with 365 nm and 470 nm light were cycled five times and the 10 datasets are stacked sequentially. Used a 2 mm path length quartz cuvette and 350 uL of solution. 365 nm light irradiation: \* There was no light irradiation during the first 10 data points (30 seconds) collected. \* The sample was irradiated with 365 nm light for 900 seconds and an absorption spectrum was collected every 3 seconds. \* After irradiation, the sample remained in the dark for 600 seconds and a spectrum was collected every 5 seconds. \* For cycles 3, 4, and 5 the sample remained in the dark for 120 seconds and a spectrum was collected every 5 seconds. 470 nm light irradiation: \* There was no light irradiation during the first 10 data points (30 seconds) collected. \* The sample was irradiated with 365 nm light for 300 seconds and an absorption spectrum was collected every 3 seconds. \* After irradiation, the sample remained in the dark for 600 seconds and a spectrum was collected every 5 seconds. \* For cycles 3, 4, and 5 the sample remained in the dark for 120 seconds and a spectrum was collected every 5 seconds. In Figure S9A/B, these periods of 365 nm, 470 nm, and no irradiation are denoted as the purple, blue, and gray shadows, respectively. The full absorbance spectra for all times provided in this dataset are available upon request. Spectra taken on home-built time-dependent UV-vis. \------------------------------------------------------------------------------------------------------------------------------------- Figure S10: Kinetics of assembly of azoEDTA with reflectin, measured by dynamic light scattering File: FigureS10.csv \* Column A: Time (min) \* Column B: Mean hydrodynamic diameter of azoEDTA-reflectin assemblies \* Column C: Standard deviation of Column B \------------------------------------------------------------------------------------------------------------------------------------- Figure S11: Ellipticity of azoEDTA-reflectin complexes evolves with time File: FigureS11.csv \* Column A: Wavelength (nm) for Column B \* Column B: Ellipticity (mdeg) of reflectin protein without azoEDTA (control) \* Column C: Wavelength (nm) for Columns D-G \* Column D: Ellipticity (mdeg) of azoEDTA-reflectin complexes after 5 min \* Column E: Ellipticity (mdeg) of azoEDTA-reflectin complexes after 70 min \* Column F: Ellipticity (mdeg) of azoEDTA-reflectin complexes after 3.6 hr \* Column G: Ellipticity (mdeg) of azoEDTA-reflectin complexes after 24 hr \------------------------------------------------------------------------------------------------------------------------------------- Figure S12A: Assembly size of 8 uM reflectin and NaCl in a 20 mM, pH 4.50 acetate buffer File: FigureS12A.csv \* Column A: Concentration of NaCl (uM) \* Column B: Mean hydrodynamic diameter (nm) of NaCl-reflectin assemblies \* Column C: Standard deviation of Column B \------------------------------------------------------------------------------------------------------------------------------------- Figure S12B: Assembly size of 8 uM reflectin and EDTA in a 20 mM, pH 4.50 acetate buffer File: FigureS12B.csv \* Column A: Concentration of EDTA (uM) \* Column B: Mean hydrodynamic diameter (nm) of EDTA-reflectin assemblies \* Column C: Standard deviation of Column B \------------------------------------------------------------------------------------------------------------------------------------- Figure S14: Circular dichroism spectroscopy controls of azoEDTA before and after irradiation with 365 nm light File: Figure14.csv \* Column A: Wavelength (nm) \* Column B: Ellipticity (mdeg) of azoEDTA prior to irradiation \* Column C: Ellipticity (mdeg) of azoEDTA after 20 minutes of irradiation with 365 nm light \------------------------------------------------------------------------------------------------------------------------------------- Figure S15A: Ellipticity of NaCl-reflectin assemblies in 20 mM, pH 4.50 acetate buffer File: FigureS15A.csv \* Column A: Wavelength (nm) \* Column B: Ellipticity (mdeg) of reflectin-only solution \* Column C: Ellipticity (mdeg) of NaCl-reflectin assemblies with 8 mM NaCl \* Column D: Ellipticity (mdeg) of NaCl-reflectin assemblies with 60 mM NaCl \* Column E: Ellipticity (mdeg) of NaCl-reflectin assemblies with 70 mM NaCl \------------------------------------------------------------------------------------------------------------------------------------- Figure S15B: Ellipticity of EDTA-reflectin assemblies in 20 mM, pH 4.50 acetate buffer File: FigureS15B.csv \* Column A: Wavelength (nm) \* Column B: Ellipticity (mdeg) of reflectin-only solution \* Column C: Ellipticity (mdeg) of EDTA-reflectin assemblies with 0.48 mM EDTA \* Column D: Ellipticity (mdeg) of EDTA-reflectin assemblies with 0.76 mM EDTA \* Column E: Ellipticity (mdeg) of EDTA-reflectin assemblies with 2.0 mM EDTA \------------------------------------------------------------------------------------------------------------------------------------- Figure S17: Precipitation experiment controls of azoEDTA and reflectin solutions File: FigureS17.csv \* Column A: Wavelength (nm) \* Column B: Absorbance of 8 uM reflectin before centrifugation \* Column C: Absorbance of 240 uM azoEDTA before centrifugation \* Column D: Absorbance of reflectin after centrifugation \* Column E: Absorbance of azoEDTA after centrifugation \------------------------------------------------------------------------------------------------------------------------------------- Figure S18: Absorbance spectra of azoEDTA-reflectin complexes, before and after centrifugation for precipitation experiments File: FigureS18.csv \* Column A: Wavelength (nm) \* Column B: Absorbance of azoEDTA-reflectin complexes before centrifugation \* Column C: Absorbance of azo-EDTA-reflectin complexes after centrifugation \------------------------------------------------------------------------------------------------------------------------------------- Figure S19: Percent of free (non-interacting) azoEDTA in the supernatant following centrifugation File: FigureS19.csv The azoEDTA-reflectin assemblies prepared with trans-azoEDTA: \* Column A: Concentration of trans-azoEDTA (uM) in azoEDTA-reflectin assemblies \* Column B: Mean fraction of trans-azoEDTA remaining in supernatant, averaged from 3 replicates \* Column C: Standard deviation of Column B The azoEDTA-reflectin assemblies prepared with cis-azoEDTA: \* Column D: Concentration of cis-azoEDTA (uM) in azoEDTA-reflectin assemblies \* Column E: Mean fraction of cis-azoEDTA remaining in supernatant, averaged from 3 replicates \* Column F: Standard deviation of Column E The absorbance spectra used for this plot can be found in the Figure 4C dataset. The equations used to calculate the fraction of azoEDTA in the supernatant can be found in the Supplementary Information. \------------------------------------------------------------------------------------------------------------------------------------- Figure S20A: Control experiment - the effect of 365 nm light irradiation on the assembly size and size distribution of reflectin File: FigureS20A.csv \* Column A: Hydrodynamic diameter (nm) \* Column B: Volume intensity distribution of the buffered reflectin sample before UV light irradiation \* Column C: Volume intensity distribution of the buffered reflectin sample after UV light irradiation \------------------------------------------------------------------------------------------------------------------------------------- Figure S20B: Control experiment - the effect of 470 nm light irradiation on the assembly size and size distribution of reflectin File: FigureS20B.csv \* Column A: Hydrodynamic diameter (nm) \* Column B: Volume intensity distribution of the buffered reflectin sample before blue light irradiation \* Column C: Volume intensity distribution of the buffered reflectin sample after blue light irradiation \------------------------------------------------------------------------------------------------------------------------------------- Figure S20C: Control experiment - the effect of 365 nm light irradiation on the ellipticity (secondary structure) of reflectin File: FigureS20C.csv \* Column A: Wavelength (nm) \* Column B: Ellipticity (mdeg) of the buffered reflectin sample before UV light irradiation \* Column C: Ellipticity (mdeg) of the buffered reflectin sample after UV light irradiation \------------------------------------------------------------------------------------------------------------------------------------- Figure S20D: Control experiment - the effect of 470 nm light irradiation on the ellipticity (secondary structure) of reflectin File: FigureS20D.csv \* Column A: Wavelength (nm) \* Column B: Ellipticity (mdeg) of the buffered reflectin sample before blue light irradiation \* Column C: Ellipticity (mdeg) of the buffered reflectin sample after blue light irradiation \------------------------------------------------------------------------------------------------------------------------------------- Figure S21A: Full absorbance spectra of 240 uM azoEDTA photoswitch before and after irradiation with 365 nm and 470 nm light File: FigureS21A.csv \* Column A: Wavelength (nm) \* Column B: Absorbance of azoEDTA prior to irradiation (stored in dark for 3 days) \* Column C: Absorbance of azoEDTA after irradiation with 365 nm light \* Column D: Absorbance of azoEDTA after irradiation with 470 nm light See Figure S9B description and Supplementary Information for details regarding irradiation. Spectra taken on home-built time-dependent UV-vis. At low wavelengths, some absorbance values were recorded as "Inf" or "NaN" due to poor detection for wavelengths &lt; 300 nm. \------------------------------------------------------------------------------------------------------------------------------------- Figure S21B: Full absorbance spectra of 240 uM azoEDTA and 8 uM reflectin before and after irradiation with 365 nm and 470 nm light File: FigureS21B.csv \* Column A: Wavelength (nm) \* Column B: Absorbance of azoEDTA-reflectin solution prior to irradiation (equilibrated for 120 minutes) \* Column C: Absorbance of azoEDTA-reflectin solution after irradiation with 365 nm light \* Column D: Absorbance of azoEDTA-reflectin solution after irradiation with 470 nm light See Figure 5B description and Supplementary Information for details regarding irradiation. Spectra taken on home-built time-dependent UV-vis. At low wavelengths, some absorbance values were recorded as "Inf" or "NaN" due to poor detection for wavelengths &lt; 300 nm. \------------------------------------------------------------------------------------------------------------------------------------- Figure S22: Time-dependent turbidity of azoEDTA-reflectin complexes prepared with 8 uM reflectin and 240 uM azoEDTA. File: FigureS22.csv \* Column A: Time (min) for Column B [0 min to 44.2 min] \* Column B: Sample turbidity at 400 nm during 365 nm irradiation cycle 1 \* Column C: Time (min) for Column D [45.7 min to 72.4 min] \* Column D: Sample turbidity at 400 nm during 470 nm irradiation cycle 1 \* Column E: Time (min) for Column F [73.9 min to 118.1 min] \* Column F: Sample turbidity at 400 nm during 365 nm irradiation cycle 2 \* Column G: Time (min) for Column H [119.6 min to 146.3 min] \* Column H: Sample turbidity at 400 nm during 470 nm irradiation cycle 2 \* Column I: Time (min) for Column J [147.8 min to 192.0 min] \* Column J: Sample turbidity at 400 nm during 365 nm irradiation cycle 3 \* Column K: Time (min) for Column L [193.5 min to 220.2 min] \* Column L: Sample turbidity at 400 nm during 365 nm irradiation cycle 3 Irradiation with 365 nm and 470 nm light were cycled three times and the 6 datasets are stacked sequentially. See Figure 5B description and Supplementary Information for details regarding irradiation and turbidity calculations. Spectra taken on home-built time-dependent UV-vis. \------------------------------------------------------------------------------------------------------------------------------------- Figure S23: Time versus percent change of mean azoEDTA-reflectin assembly size over 2 irradiation cycles File: FigureS23.csv \* Column A: Time (min) \* Column B: Fraction of size change when DLS measurement is taken immediately following irradiation \* Column C: Fraction of size change when DLS measurement is taken 15 minutes following irradiation \------------------------------------------------------------------------------------------------------------------------------------- Figure S24: Time versus mean hydrodynamic diameter of azoEDTA-reflectin assemblies over 2 irradiation cycles, starting from cis isomer File: FigureS24.csv \* Column A: Time (min) \* Column B: Mean hydrodynamic diameter (nm) of azoEDTA-reflectin assemblies when irradiation cycle starts with cis-azoEDTA \------------------------------------------------------------------------------------------------------------------------------------- Figure S25: Count rate versus hydrodynamic diameter of azoEDTA-reflectin assemblies - valid for diameters &lt; 100 nm File: FigureS25.csv \* Column A: Hydrodynamic diameter (nm) for 95% confidence interval bounds, Columns B and C \* Column B: Lower bound of confidence interval for count rate (kcps) \* Column C: Upper bound of confidence interval for coutn rate (kcps) \* Column D: Hydrodynamic diameter (nm) for Column E \* Column E: Count rate (kcps) from biexponential model \* Column F: Hydrodynamic diameter (nm) [real data] for measured azoEDTA-reflectin samples \* Column G: Count rate (kcps) [real data] for the measured azoEDTA-reflectin samples \------------------------------------------------------------------------------------------------------------------------------------- Figure S26: Time versus count rate during DLS measurements of azoEDTA-reflectin assemblies over 2 irradiation cycles File: FigureS26.csv \* Column A: Time (min) \* Column B: Count rate (kcps) of azoEDTA-reflectin assemblies \------------------------------------------------------------------------------------------------------------------------------------- Figure S27: Proton NMR of compound 6 File: FigureS27.csv \* Column A: Chemical shift (ppm) \* Column B: Signal intensity \------------------------------------------------------------------------------------------------------------------------------------- Figure S28: Carbon NMR of compound 6 File: FigureS28.csv \* Column A: Chemical shift (ppm) \* Column B: Signal intensity \------------------------------------------------------------------------------------------------------------------------------------- Figure S29: Proton NMR of compound 7 File: FigureS29.csv \* Column A: Chemical shift (ppm) \* Column B: Signal intensity \------------------------------------------------------------------------------------------------------------------------------------- Figure S30: Carbon NMR of compound 7 File: FigureS30.csv \* Column A: Chemical shift (ppm) \* Column B: Signal intensity \-------------------------------------------------------------------------------------------------------------------------------------