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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.
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