When Monomers Are Preferred: A Strategy for the Identification and Disruption of Weakly Oligomerized Proteins  Yufeng Tong, David Hughes, Lisa Placanica, Matthias Buck  Structure  Volume 13, Issue 1, Pages 7-15 (January 2005) DOI: 10.1016/j.str.2004.10.018 Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 1 Wild-Type Plexin-B1 RBD at Different Protein Concentrations (A) Retention volume of plexin-B1 RBD on sephadex-75 gel filtration column at different concentrations, measured by UV absorbance at 280 nm, 276K. (B) 15N-1H HSQC of wild-type plexin RBD at 1.4 mM, 298 K. (C) 15N-1H HSQC of wild-type plexin RBD at 10 μM, 298 K. Each signal in the displayed regions arises from a different amide. Structure 2005 13, 7-15DOI: (10.1016/j.str.2004.10.018) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 2 NMR Backbone Assignment and Sequence Analysis of the Plexin-B1 RBD (A) Hydrophobicity scale (Eisenberg et al., 1984) as a measure of polarity and Jnet secondary structure prediction (Cuff and Barton, 2000) of the plexin RBD. For the hydrophobicity calculation, a window size of 5 was chosen. A lower value indicates a locally more polar region. Other hydrophobicity scales give nearly identical results. “Jpred” indicates the consensus prediction results of the Jnet method, where H stands for helical and E stands for extended secondary structure. “Jnet Rel” is the prediction accuracy, scaling from 0 to 9. Residues assigned initially from spectra of the dimer are shadowed. Prolines are indicated by arrows. A yellow line and a green line are drawn at hydrophobicity values of 0.5 and 0.7, respectively. Sites chosen for mutagenesis are labeled with +. (B) Alignment of the RBD of plexin-B1 with members of the protein family from different species. Nonconserved inserts of 26 residues in R. norvegicus plexin B3 and 7 residues in D. melanogaster plexin B, indicated by black arrows, are omitted in the plot for clarity. Conserved sites with five or more residues identical in the alignment are shadowed green in the consensus. Structure 2005 13, 7-15DOI: (10.1016/j.str.2004.10.018) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 3 Association Status of Representative Mutants Retention volumes of (A) dimeric and (B) monomeric mutants on gel filtration shown in comparison with the retention volume profile of wild-type plexin-B1 RBD. Structure 2005 13, 7-15DOI: (10.1016/j.str.2004.10.018) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 4 Rac1 Binding Activity of Plexin-B1 RBDs Pull-down assay of untagged wild-type plexin-B1 RBD, W1830F, H1838S, and Y1839S with GST-tagged Rac1.Q61L show different binding activities. Star (*) and arrow (→) indicate the position of GST-Rac1.Q61L and the plexin RBDs, respectively. Structure 2005 13, 7-15DOI: (10.1016/j.str.2004.10.018) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 5 Part of the 15N-1H HSQC Spectra of Plexin-B1 RBD Mutants at 298 K (A) Spectrum of RBD-W1830F shows nearly identical distribution of peaks to that of the wild-type RBD. (B) Spectrum of RBD-H1838S is also well dispersed but significantly different from that of RBD-W1830F. (C) Spectrum of RBD-Y1839E shows a population of unfolded protein. (D) Spectrum of RBD-N1834D shows a coexistence of two conformations. Structure 2005 13, 7-15DOI: (10.1016/j.str.2004.10.018) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 6 Identification of the Dimer Interface of the Plexin-B1 RBD The change of amide signal intensities between HSQC spectra at two different concentrations of wild-type plexin-B1 RBD at 298 K are plotted against protein sequence. Intensity at 0.2 mM was divided by that of the corresponding signals at 10 μM protein concentration and normalized to the intensity ratio of the C-terminal amide Q1862, which is not affected by the association. Structure 2005 13, 7-15DOI: (10.1016/j.str.2004.10.018) Copyright © 2005 Elsevier Ltd Terms and Conditions