Identification of RKIP Ligands Figure 1. Overlay of 1H,15N-HSQC spectra of RKIP in the absence (black) and presence (red) of a potential ligand. (A) PE (10 mM) does not result in any significant chemical shift perturbations. (B) 1H,15N-HSQC spectra of RKIP taken at pH 6.0 (red) and at pH 7.4 (black) showing a large number of missing peaks at pH 6.0. In all the panels, red contours are plotted over black contours, and thus a black cross peak appears only when its corresponding peak drawn in red is either shifted or missing. RKIP chemical shift perturbations for amide proton resonances by PE (10 mM) (C) or DHPE (4.5 mM) (D) at pH 7.4 plotted as a function of residue number. Secondary structural elements are depicted at the top with beta-sheet and alpha-helices represented by closed and open boxes, respectively. Gray boxes indicate the position of residues that form the ligand-binding pocket. (E) RKIP chemical shift perturbations by PE (100 mM) at pH 6.0. Note that panel E is plotted on a smaller vertical scale than that for C and D. doi:10.1371/journal.pone.0010479.g001 the bovine RKIP/PE complex [17]. Because the rat and bovine proteins are highly homologous, particularly among the residues forming the pocket, it is unlikely that these proteins have distinctly different ligand-binding properties. The RKIP/PE complex was crystallized at pH 4.6, which is much lower than the pH used in our solution studies (pH 7.4). The pocket contains a conserved His residue (His-85 in the bovine RKIP numbering), and the pKa of His is typically between 6.0-7.0 [28]. These observations led us to hypothesize that pH may affect the structure and function of the RKIP pocket. To investigate the effect of pH, we performed pH titration of RKIP monitoring the amide proton chemical shifts with the 15N-HSQC spectrum. PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10479