Supplementary Figure S1. Amino Acid Sequences of Prs2in and Prs2ic. Sequences of Prs2in

Supplementary Figure S1. Amino acid sequences of PrS2IN and PrS2IC. Sequences of PrS2IN and PrS2IC used in this study are shown. Junction amino acids (SGV; residue 228-230) in PrS2IC are indicated by red letters.

Supplementary Figure S2. MALDI-TOF mass spectrum of PrS2-RbfA (15N) generated by PSTI technology. The measured mass of PrS2-RbfA (15N) is 35118 Da (calculated mass = 35092 Da). BSA (mass = 66431 Da) was used for standard.

Supplementary Figure S3. The differences of HN and 15N chemical shifts between RbfAD25 and PrS2-RbfAD25 (15N). The differences were calculated with the formula Dd=√[(DHN)2+0.2(D15N)2]/2 and the values are normalized. The dot line indicates Dd = 0.1 (ppm).

Supplementary Figure S4. In vitro protein ligation of PrSIN and PrS2IC-RbfA. SDS-PAGE analysis of protein ligation of PrSIN and PrS2IC-RbfA. Protein ligation was performed at 25 oC and the efficiency of the reaction was examined by SDS-PAGE. M, Molecular weight marker; Lanes: 1, Soluble fraction of PrSIN; 2, Soluble fraction of PrS2IC-RbfA; 3, 0 hr of protein ligation; 4, 16 hr of protein ligation; 5, purified PrS-RbfA (15N, 13C).

Supplementary Figure S5. [1H-15N]-TROSY HSQC spectrum of PrS-RbfA (15N, 13C) and comparison with PrS2-RbfA (15N). The [1H-15N]-TROSY HSQC spectrum of segmentally-labeled PrS-RbfA (15N, 13C) (black peaks) on the spectrum PrS2-RbfA (15N) (magenta peaks). Both spectra were recorded at 800 MHz and temperature of 35 oC.

Supplementary Figure S6. Comparison of N-H projection CBCA(CO)NH TR-NMR spectrum of PrS2-RbfA (15N, 13C) and PrS-RbfA (15N, 13C). The CBCA(CO)NH spectrum of segmentally-labeled PrS2-RbfA (15N, 13C) (magenda peaks) is overlayed onto the spectrum PrS-RbfA (15N, 13C) (black peaks). Both proteins were prepared at 0.5 mM, and were recorded at 600 MHz and temperature of 35 oC. These data document the more favorable properties of PrS compared with PrS2 as an “invisible SET”.

Supplementary Figure S7. TR-NMR connectivity map for RbfAD25 and full-length RbfA. Intra-residue and sequential connections derived from TR-NMR spectra and used to determine backbone resonance assignments for RbfAD25) and for full-length RbfA, along with the residue number. Red horizontal lines indicate intra-residue (i) and yellow sequential (s), respectively. Also shown are site-specific 1H-15N-HetNOE and Ca Chemical Shift Index data, along with the secondary structure observed in the 3D structure of RbfAD25.

Supplementary Figure S8. CS-Rosetta results. Chemical shift rescored Rosetta energy vs. GDT-TS score relative to the lowest energy structure.

Supplementary Figure S9. [1H-15N]-TROSY HSQC spectrum of PrS2-RbfA (15N). Uniformly 15N-enriched PrS2IC-RbfA was expressed using the condensed-phase SPP system in 20-fold condensed isotope-enriched medium. The expressed protein was fused by protein splicing with non-isotope labeled PrS2IN to generate PrS2-RbfA (15N). The [1H-15N]-TROSY HSQC spectrum of the spliced product was obtained at 800 MHz, 35 oC and pH 4.0.