SUPPLEMENTARY INFORMATION. Tolerance of a knotted near infrared fluorescent protein to random circular permutation

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1 SUPPLEMENTARY INFORMATION Tolerance of a knotted near infrared fluorescent protein to random circular permutation Naresh Pandey 1,3, Brianna E. Kuypers 2,4, Barbara Nassif 1, Emily E. Thomas 1,3, Razan N. Alnahhas 1,3, Laura Segatori 1, 4,5, and Jonathan J. Silberg 1,5 * Author affiliations: 1 Department of Biosciences, Rice University, Houston, Texas 77005, United States 2 Systems, Synthetic, and Physical Biology Graduate Program, Rice University, Houston, Texas 77005, United States 3 Biochemistry and Cell Biology Graduate Program, Rice University, Houston, Texas 77005, United States 4 Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas 77005, United States 5 Department of Bioengineering, Rice University, Houston, Texas 77005, United States *To whom correspondence should be addressed: Tel: ; joff@rice.edu 1

2 Permuteposon (23 C) Permuteposon (37 C) pbad (37 C) Variant λ ex λ em σ λ ex λ em σ λ ex λ em nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 0 nd σ Table S1. Emission and excitation maxima of circularly permuted irfp. To determine the wavelengths that yield maximum excitation (λ ex ) and emission (λ em ), spectra were measured using E. coli XL1 Blue expressing each permuted irfp from permuteposon P4 or a pbad vector that expresses each variant with a N-terminal His tag. Each protein is named based on the irfp residue at the NH 2 -terminus, and fluorescence signals are reported as the number of standard deviation (σ) of each variant over that observed with cells harboring a vector that lacks the irfp gene. Cells expressing wild-type irfp yielded maximal emission upon excitation at 694 nm and maximal excitation when emission was monitored at 715 nm. 2

3 Figure S1. Permuteposon used for library construction. (A) Permuteposon P4 was created by incorporating mutations (yellow highlights) into the R2R1 transposase recognition sequence of P1 to generates a RBS. This modified R2R1 leads to translation initiation closer to the end of the permuteposons and adds only two residues to permuted variants. P4 also differs in its transcriptional regulation from P1. A tac promoter (P Tac ) with a lac operator is used in P4 to regulate transcription of permuted proteins. (B) Quality assessment of the library. When digested with NotI, the final library yields a single band that corresponds to the molecular weight of P4 (1911 bp) and the irfp gene (993 bp). When digested with NotI and NcoI, a smear is observed, consistent with the library containing an ensemble of permuted irfp. 3

4 Figure S2. Effect of BV concentration on permuted protein fluorescence. The fluorescence (λ ex = 690 nm; λ em = 715 nm) of E. coli XL1 Blue expressing different circularly permuted irfp was measured in cells grown in medium supplemented with varying BV concentrations (0, 5, 10, 20, 40, and 80 µm) and 1 mm arabinose. Fluorescence that had been normalized to the maximum signal obtained with each variant was fit to a hyperbolic saturation function to obtain the apparent affinity (K M ) within whole cells. 4

5 Figure S3. Fluorescence of affinity-tagged irfp. (A) The fluorescence (λ ex = 690 nm; λ em = 715 nm) of E. coli XL1 Blue expressing different circularly permuted irfp with His tags was measured at 37 C and normalized to cell density. The signal from each variant is reported as the fraction of the signal obtained with the variant displaying the highest fluorescence. (B) The ratio of fluorescence measured for circularly permuted irfp with a His tag (+tag) to the same variant lacking a tag (-tag). The dashed line represents the ratio expected if expression does not vary with tag addition. Error bars represent ±1σ from three or more experiments. 5

6 Figure S4. Immunoblot analysis of protein expression. E. coli XL1-Blue transformed with pbad vectors that express circularly permuted irfp were subjected to Western immunoblot analysis using an anti-(his) 6 antibody. To establish the relative expression of each permuted variant, cells expressing unmodified irfp was included as a control. Cells transformed with a pbad vector lacking a BphP were included as a negative control. For each circularly permuted variant (8, 11, 12, 101, 102, 123, 127, 129, 133, 134, 136, 142, 198), experiments were performed using three samples derived from distinct colonies transformed with the same plasmid. All blots contained unmodified irfp to allow for comparisons between different blots. 6

7 Figure S5. Absorbance of recombinant permuted irfp. Data acquired using purified proteins yielded similar absorbance peaks as native irfp. A comparison of the ratio of the absorbance arising from BV (391 nm) and total protein (280 nm) yielded ratios for each permuted variant ( ), which were similar to unmodified irfp (0.85). 7

8 Figure S6. Flow cytometry analysis of tissue culture fluorescence. (A) Green and nearinfrared fluorescence emission of HeLa cells transiently transfected with a pair of vectors that express GFP and select circularly permuted irfp variants. For each sample, five independent measurements were performed on two different days. (B) Western immunoblot analysis of HA-tagged near-infrared fluorescent proteins is compared with the GFP transfection control and α-tubulin loading control. (C) The GFPnormalized near-infrared signal from cells was normalized to the amount of each nearinfrared fluorescent protein, which had been normalized to the tubulin loading control. Error bars represent ±1σ. Protein expression levels were estimated by including all bands that maintain the HA tag because our purpose was to normalize the cell fluorescence signal by the total protein expressed. It is not known where the cleavage sites occur or if biliverdin binding is maintained in all products. 8