For more detailed studies of pr-E binding, we performed surface plasmon resonance (SPR) assays using our various forms of recombinant E protein with immobilized pr peptide. Compared to the pull-down assay, SPR can detect low levels of protein-protein interactions as binding is detected in real time and does not require removal of unbound E. The E9 protein is a dimer at neutral pH and dissociates to monomers at low pH [30] . When SPR was performed with E9 protein buffered at pH 8.0 there was very low binding (low signal response) (Fig. 2B ). As the buffer pH was Figure 2 . Pr peptide binds DENV E proteins in a pH-dependent manner. A) Pull-down of DI/II protein by pr. DI/II was incubated with sepharose beads conjugated with pr peptide or BSA at the indicated pH for 1 h at room temperature. As indicated, reactions contained a 2:1 molar excess of mAb 4G2 to the E fusion loop or mAb to the ST tag (con.). Input lanes show an aliquot representing 20% of the reaction prior to pull-down. (Panels B-D) SPR analysis of pr-E binding. Pr peptide was immobilized on a CM5 sensor chip, and DENV2 E9 (B), DI/II (C) or SFV DI/II proteins (D) were flowed over the chip at concentrations of 1.2 mM in buffers of the indicated pH for 300 s, followed by injection of protein-free buffer at the same pH. Data are a representative example of two independent experiments. doi:10.1371/journal.ppat.1001157.g002 decreased, the signal gradually increased, with maximal response observed at ,pH 6.25 and no further increase at pH 6.0. A rapid decrease in signal was observed when the samples were shifted to protein-free buffer, indicating rapid dissociation of the pr-E interaction. Similar results were obtained using monomeric DI/ II, with the lowest binding at pH 8.0, highest binding at pH 6.25, and a slight decrease at pH 6.0 (Fig. 2C) . Thus, the dimeric E9 and monomeric E DI/II proteins bound pr peptide with similar pHdependence. Binding to pr was specific, as little interaction was observed using the structurally similar E1 DI/II protein of SFV (Fig. 2D ). In addition, binding of DENV E DI/II protein to pr was inhibited by preincubation with mAb 4G2 against the fusion loop (molar ratio 1:1) (data not shown). Determination of the affinity of pr-E binding was not performed as the data did not fit to a simple Langmuir model of 1:1 binding, presumably because of E protein aggregation at low pH.