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SUPPLEMENTARY METHODS Plasmid construction. DNA coding for full-length ubiquitin (amino acids 1-76) was amplified from pET3a-ubiquitin (a gift from C. Pickart, Johns Hopkins University) using the oligonucleotides TTTTTTCCATGGCAATCTTCGTCAAGACGTTAACCGG-3' 5'- and 5'- CCCAAGCTTTCAACCACCTCTTAGTCTTAAGACAAGATGTAAGG-3'. The gene was ligated into pBAD-HisA (Invitrogen) using the NcoI and HindIII linker sites. The resulting plasmid, pBAD-Ubq, expresses Ubiquitin from the araBAD promoter, which is induced by L-arabinose. pBAD-Ubq confers ampicillin resistance and contains a Col E1 replication origin and the araC gene, which codes for a transcription regulator. DNA coding for the ataxin-3 peptide, AUIM (MLDEDEEDLQRALALSRQEIDMEDEEAD), was synthesized and ligated into pRSF-1b (Novagen) using the NcoI and SalI linker sites. Mutation of AUIM Ser16 to Ala (SA16) was accomplished using the Quick Change kit (Stratagene). DNA coding for full-length mouse STAM2 (amino acids 1-523) was amplified from pGEX-STAM2 (a gift from H. Stenmark, Institute for Cancer Research, Norway) using the oligonucleotides 5'-GTAGCAGTCGACCTACAGGAGAGG-3' and 5'-TTTTTTCCATGGCTCTGTTCACTGC-3' and ligated into pRSF-1b (Novagen) using the SalI and NcoI linker sites. The resulting plasmids, pRSFAUIM, pRSF-AUIM(SA16), and pRSF-STAM2, express AUIM, AUIM(SA16), and STAM2, respectively, from a T7 promoter/lac operator, which is induced by isopropyl -D-thiogalactoside (IPTG). These plasmids confer kanamycin resistance and contain an RSF replication origin and the lacI gene, which codes for Lac repressor. Expression of [U-15N]-Ubiquitin. E.coli Rosetta(DE3) (Novagen) was cotransformed with plasmids pBAD-Ubq and either pRSF-AUIM, pRSF- AUIM(SA16) or pRSF-STAM2. This strain contains the pRARE plasmid, which over-expresses tRNAs for Arg, Leu, and Pro codons rarely used by E. coli. pRARE is compatible with both pBAD-HisA and pRSF-1b. Cells were grown overnight at 37 oC in Luria-Bertani medium (LB) supplemented with 100 mg/L ampicillin and 35 mg/L kanamycin, to an OD600 of 1.0-1.2. Growing the culture to a higher OD results in the loss of the ampicillin resistant plasmid by the bacteria (since ampicillin is secreted into the growth medium) and the subsequent inability to express protein. The cells were washed once with minimal medium (M9) salts (40 mL per 20 mL cell culture) and re-suspended to an OD600 of 0.5-0.6 in 200 mL of M9 containing [U-15N] ammonium chloride (0.7 g/L) and glycerol (4 mL/L) as the sole nitrogen and carbon sources, 100 mg/L ampicillin and 35 mg/L kanamycin. The culture was incubated at 37 oC for 10 minutes and ubiquitin expression was induced by adding 1/100 th culture volume of 20% w/v of L-arabinose. Induction was allowed to proceed for 3 hours; a second aliquot of 1/200th culture volume of L-arabinose was added after 2 hours of induction. The OD600 of the culture approximately doubled over this time. Following the first induction, a 50 mL sample was taken, the cells were centrifuged, washed three times with 50 mL of 10 mM potassium phosphate buffer [pH 7] and stored at -80 oC for subsequent NMR analysis. Expression of AUIM, AUIM(SA16) and STAM2. The remaining culture was centrifuged, washed once with 200 mL of M9 salts and a sufficient number of cells containing [15N]-labeled ubiquitin were re-suspended to yield an OD600 of 0.5-0.6 in 200 mL of M9 containing ammonium chloride (0.7 g/L), 2 g/L of dextrose, 2 g/L casamino acids, 100 mg/L ampicillin and 35 mg/L kanamycin. The casamino acids minimized proteolytic degradation of the labeled ubiquitin by providing an additional carbon and nitrogen source for on-going protein overexpression. The culture was incubated at 37 oC for 10 minutes and 1/1000th culture volume of 0.5 M IPTG was added. The expression of AUIM, AUIM(SA16) or STAM2 was allowed to proceed for 3 hours. Residual expression of labeled protein off the araBAD promoter is further suppressed by the presence of dextrose in the second M9 medium, preventing dilution of the labeled protein. 50 mL samples were taken at various times following induction. The OD600 of the culture typically increased by less than 20% over 3 hours of induction. Cells were centrifuged, washed three times with 50 mL of 10 mM potassium phosphate buffer [pH 7] and stored at - °C for subsequent NMR analysis. It is important to note that successful sequential expression can only be done when the T7-based promoter is the last to be induced. When the over- expression and labeling protocol is reversed, that is, the unlabeled ligand encoded by the pRSF plasmid is expressed to a high concentration first, followed by over-expression of the isotope labeled target by the pBAD plasmid, there is significant contamination of the target protein NMR spectra by peaks originating from the labeled ligand. Once over-expression using the T7 promoter is induced, it can not be effectively stopped by simply removing the inducer. We found that the ligand protein encoded by pRSF plasmid was produced continuously, even up to 4 hours after IPTG was removed, presumably by the large pool of T7 RNA polymerase present. However, by switching expression vectors, either protein can be expressed and labeled first or both proteins can be labeled using different labeling strategies. STINT-NMR can be performed in different ways to achieve the ratios of interactor to target necessary for a complete structural titration. This method can be combined with in vitro methodology, for example, by exogenously adding a small interacting molecule to cell cultures expressing an endogenously labeled protein. The most comprehensive experimental protocol entails a matrix of samples wherein the target molecule is expressed to different concentrations and a titration curve generated for each concentration of labeled target. These titrations lack the precision of typical binding isoterms due to the variable levels of expression inherent when using living cells and are, therefore, largely qualitative. Because the same structural endpoints are achieved, quantitating protein concentrations is needed only to generate binding curves to estimate binding affinities; approximations of this type could be accomplished using western blots to quantitate total cellular protein levels. Since we are measuring interactions within the confines of a single cell, and the effective concentration of interactor proteins varies from 0.1-10 mM, we can identify potein-protein interactions that span a broad range of binding affinities, from micromolar to millimolar. NMR spectroscopy. Frozen [U- 15N] labeled cells were resuspended in 0.5 mL of NMR buffer (10 mM potassium phosphate, pH 7.0, 90%/10% H2O/D2O) and transferred to the NMR tube. Due to the high cell density inside the NMR tube, re-suspended cells do not sediment. To rule out the possibility that the visible NMR spectrum was due to intercellular proteins, we sedimented the cells from the NMR sample and acquired 1H{15N}-HSQC spectrum of the resultant supernatant. No protein NMR signal was visible above the noise level (Supplementary Fig. 6). In addition, SDS-PAGE of the cells after 4 hours in the NMR tube showed minimal protein degradation (Supplementary Fig. 1). We also collected 1H{15N}-HSQC spectrum of the E. coli cells grown on [U-15N] labeled medium without protein over-expression (Supplementary Fig. 7). The resultant spectrum exhibited 19 sharp peaks located between 7.5 ppm and 8.5 ppm corresponding to small metabolites of [U-15N] ammonium chloride. All NMR experiments were acquired on Bruker Avance 700 MHz NMR spectrometer equipped with a cryoprobe. We used a watergate version of 1H{15N}-HSQC experiment. 1H{15N}-edited HSQC data were recorded with 16 transients as 512{128} complex points, apodized with a squared cosine-bell window function and zero-filled to 1k{512) points prior to Fourier transformation. Acquisition of each NMR spectrum took ~1 hour. The corresponding sweep widths were 12 and 35 ppm in 1H and 15N dimensions, respectively. In in-cell binding titration experiments, we measured the change in the chemical shifts of amide nitrogens and covalently attached amide proton according to the equation: (N2 /25 H2 ) , where H (N ) represents a change in hydrogen and nitrogen chemical shifts.