Supporting Information File 3 (SI-3)
Surface Plasmon Resonance Analysis
Proteins used in the SPR analyses were subjected to purification steps in addition to those
described in SI-2 (Protein Purification). The wild-type and mutant Ec-YmdB and Ec-RNase III
proteins, purified as described in SI-2, were subjected to size exclusion chromatography using a
Superdex-750/300 GL column on an Akta-FPLC system. Prior to use the column was
extensively washed with a 20% ethanol/water solution, then extensively washed with doubledistilled water. The column was equilibrated with running buffer (500 mM NaCl, 30 mM TrisHCl, pH 8.0) using the preset program, until the UV absorbance (280 nm) approached zero. The
running buffer equilibration was continued for an additional 20 min. Proteins were dialyzed
against running buffer then injected onto the column. Fractions (1 ml fraction volumes, collected
at a rate of 0.5 ml/min) were collected in separate tubes and the protein content analyzed by
SDS-PAGE. The protein fractions were combined and stored at -20ºC in 50% (v/v) glycerol.
The SPR analyses required removal of the N-terminal H6-tag from Ec-RNase III. H6-EcRNase III (~300 μg) was treated with recombinant biotinylated bovine thrombin (6 units,
Novagen,) for 48 hr at room temperature in a 1 ml reaction with 250 mM NaCl and 30 mM TrisHCl (pH 8.4) buffer. The biotinylated thrombin was removed by treatment with streptavidinagarose beads (32 μl added per unit of thrombin used). To remove the released H6-tag (~2 kDa),
the protein solution was dialyzed against buffer C (500 mM NaCl, 60 mM Tris-HCl (pH 8.0), 1
mM EDTA, and 1 mM DTT) using Spectra/Por dialysis membranes (3500 MW cutoff). In this
step the H6-tag passes through the membrane while the Ec-RNase III is retained. Next, the
dialyzed solution was passed through a Qiagen-Ni-NTA column to remove residual H6-tag, as
well as any remaining unreacted H6-tagged protein. The eluate was collected and stored in the
presence of 50% glycerol at -20ºC.
The Ec-YmdB-Ec-RNase III interaction was quantitatively analyzed using a Biacore 2000
System (GE HealthCare). A Sensor Chip NTA was used, which is composed of
carboxymethylated dextran with covalently attached nitrilotriaacetic acid (NTA) groups. The
NTA molecules bind Ni2+ ions, retaining free coordination sites that can bind H6-tagged protein,
providing a stably-affixed protein (“bait”). A 0.5 mM NiCl2 solution was used to charge the
sensor chip surface. To prepare protein for analysis, the storage buffer was exchanged for SPR
running buffer (150 mM NaCl, 30 mM Tris-HCl, pH 7.5) using an Amicon Ultracentrifugal filter
(EMD-Millipore) immediately before loading the sample into the SPR unit. The H6-tagged EcYmdB (wt or R40A mutant) was injected, providing approximately 300 Response Units (RU).
Ec-RNase III (wt or D128A mutant) was then injected as the analyte (“prey”) at specified
concentrations. An additional response signal was obtained, indicating complex formation, with
the subsequent injection of binding buffer causing a progressive decrease in signal, indicative of
complex dissociation. Following each analysis, the sensor chip was regenerated by injecting a
solution of 350 mM EDTA. The NTA chip could be regenerated and used up to ~50 times, with
reliable results. The chip was stored at 4ºC when not in use, to minimize deterioration. The
association rate constant (ka) and dissociation rate constant (kd) were obtained from the RU
curves using the BIAevaluation 4.1.1.exe program, from which the equilibrium dissociation
constant (KD) was calculated. Igor6 software was used to improve the output sensogram tracings.