SUPPLEMENTARY INFORMATION

Size: px

Start display at page:

Download "SUPPLEMENTARY INFORMATION"

Neal Stevens
5 years ago
Views:

100 ng of YUH was added to 350 l of 1 mm 15 N-labeled ubiquitin AAA -CPP TAT in PBS at ph 7.4.

1 Figure S1. Cleavage of uniquitin AAA -CPP TAT in vitro and in cells. a, b. In vitro two-dimensional 1 H- 15 N correlation spectrum of ubiquitin AAA -CPP TAT before (a) and after (b) Yeast Ubiquitin Hydrolase (YUH) treatment. 100 ng of YUH was added to 350 l of 1 mm 15 N-labeled ubiquitin AAA -CPP TAT in PBS at ph 7.4. After incubation at 37 C for 15 hrs, the two-dimensional 1 H- 15 N correlation spectrum of the reaction mixture was acquired at 37 C. The cross-peaks of Gly 76 of ubiquitin AAA and C-terminal Arg 88 of CPP TAT are marked in circles. We made the assignment of Arg 88 of CPP TAT, based on the absence of the cross-peak in the spectrum of 1

2 ubiquitin AAA -CPP TAT harboring an R88A mutation (data not shown). The absence of cross-peak of Arg 88 in the spectrum of HeLa cells treated with ubiquitin AAA -CPP TAT (Figure 1a) indicates that the clipped CPP peptide was not observed in the in-cell NMR spectrum. c, Intracellular cleavage of Alexa-488-labeled ubiquitin AAA - 75 GG 76 -CPP TAT and ubiquitin AAA - 75 AA 76 -CPP TAT. The labeled proteins were prepared as described in Methods. HeLa cells were incubated with the labeled proteins in the presence of pyrenebutyrate. After cell disruption, proteins in the cell lysates were analyzed by 17% SDS-PAGE and fluorimaging. The fluorimage was obtained by using Fluor Imager 595 and ImageQuant software (Molecular Dynamics) with an excitation at 488 nm and a 530 DF 30 emission filter. Lane 1, lysate of cells treated with ubiqutin AAA - 75 GG 76 -CPP TAT ; 2, lysate of cells treated with ubiqutin AAA - 75 AA 76 -CPP TAT ; 3, ubiqutin AAA - 75 GG 76 -CPP TAT after incubation with YUH in vitro; 4, intact ubiqutin AAA - 75 GG 76 -CPP TAT. To prepare cleaved ubiquitin AAA - 75 GG 76 -CPP TAT (lane 3), 28.8 ng of the protein was incubated with 58.8 ng of Yeast Ubiquitin Hydrolase in PBS (ph 7.4) for 30 min at 37 C. 2

3 Figure S2. Assessment of protein leakage during in-cell NMR experiment. It is important to verify that the NMR signals derive from intracellular proteins, and that the contribution from proteins in the extracellular fluid is negligible. To evaluate the contribution from 15 N-labelled protein that leaked from the cells during the in-cell NMR experiment using 15 N-labeled ubiquitin AAA -CPP TAT, we measured the one-dimensional 1 H-{ 15 N} SOFAST-HMQC spectrum of the spent medium (a). This spectrum was then compared with that of the cell suspension (b). The number of scans used to acquire the spectrum of the spent medium was increased to compensate for the dilution factor of the supernatant sample (e.g. if the spent medium was twice diluted for the measurement, the number of scans was four times greater than that for the corresponding cell suspension). The spent medium was diluted with an equal volume of PBS, ph 7.3 (i.e., dilution 3

4 factor of 2). The number of scans was 2048 for spectrum (a) and 512 for spectrum (b). The markedly low signal intensity observed for spectrum (a) demonstrates that leaked protein contributes a negligible amount to the in-cell spectrum (b). Shown in (c) is the spectrum of the transduced protein recovered in cell lysate collected by cell disruption. The number of scans was 512. In each in-cell NMR experiment described in this paper, the NMR signals observed for the spent medium were markedly weaker than those for the cell suspension, indicating that the signal from extracellular 15 N proteins was negligible in our in-cell NMR experiments. 4

5 Figure S3. In-cell 1 H- 15 N correlation NMR spectrum of monkey COS-7 cells treated with ubiquitin AAA -CPP TAT. Shown is the spectrum of COS-7 cells treated with ubiquitin AAA -CPP TAT. The cross peak at a position corresponding to the C-terminal Gly76 of mature ubiquitin is marked by a circle. The experimental procedure is essentially the same as that used to obtain the in-cell NMR spectrum of ubiquitin AAA in HeLa cells shown in Fig. 1a. For the generation of spectrum, data from two experiments, each with a measurement time of 3 hrs, were added and processed. 5

6 Figure S4. In-cell 1 H- 15 N correlation NMR spectrum of HeLa cells treated with ubiquitin-cpp TAT. The cross peak at a position corresponding to the C-terminal Gly 76 of mature ubiquitin is marked by a circle. The experimental procedure is essentially the same as that used to obtain the in-cell NMR spectrum of ubiquitin AAA shown in Fig. 1a. The measurement time was 3 hrs. 6

7 Figure S5. Measurement of hydrogen exchange rates from the in vitro spectra of ubiquitin and ubiquitin AAA recovered in the lysate. a, Two-dimensional 1 H- 15 N correlation spectra of lysates prepared from HeLa cells treated with 15 N-labeled deuterated ubiquitin-cpp TAT. The lysates were collected by disruption of the cells at 30, 60, 120 and 210 min after the start of protein transduction. 7

8 See text for the experimental procedures. The cross-peaks of the main chain amides of Val 5, Leu 15, Lys 27 and Ile 30, which are highly protected in the folded state, are indicated. As the exchange rate is markedly low at ph 5, the protonation states of protected amides in ubiquitin at the time of cell disruption are preserved during the NMR measurement (see Methods). On the other hand, the measurement time of the in-cell spectrum (Fig. 4a) is too long for quantitative analysis of the exchange rate in cells, because the protonation levels largely increase in the cells during the measurement. b, Build-up of the cross peaks of the amides of Val 5, Leu 15, Lys 27 and Ile 30 observed in spectra of ubiquitin AAA recovered in lysates collected at various times after the start of protein transduction (filled symbols). Hydrogen exchanges of the amides in vitro at ph 7.4 are also shown (open symbols). Error bars indicate standard deviations. 8

Nature Methods: doi: /nmeth Supplementary Figure 1

Nature Methods: doi: /nmeth Supplementary Figure 1 Supplementary Figure 1 Subtiligase-catalyzed ligations with ubiquitin thioesters and 10-mer biotinylated peptides. (a) General scheme for ligations between ubiquitin thioesters and 10-mer, biotinylated