Head-to-Tail Dimerization and Organogelating Properties of Click Peptidomimetics

Click triazole-based oligopeptides <b>1</b>–<b>3</b> were found to self-dimerize (<i>K</i>_dim ≈ 10–680 M^–1) in a head-to-tail fashion based on ¹H variable concentration, 2D, and H/D exchange NMR, VPO, CD, FT-IR studies and Gaussian 03 simulations. The dimerization constant <i>K</i>_dim was shown to increase with increasing number of the amino acid units. Within the same oligomeric series, the <i>K</i>_dim value is strongly affected by the size of the <i>C</i>-terminal end group. The tripeptides <b>2</b> are also excellent organogelators of aromatic solvents

Head-to-Tail Dimerization and Organogelating Properties of Click Peptidomimetics

Click triazole-based oligopeptides <b>1</b>–<b>3</b> were found to self-dimerize (<i>K</i>_dim ≈ 10–680 M^–1) in a head-to-tail fashion based on ¹H variable concentration, 2D, and H/D exchange NMR, VPO, CD, FT-IR studies and Gaussian 03 simulations. The dimerization constant <i>K</i>_dim was shown to increase with increasing number of the amino acid units. Within the same oligomeric series, the <i>K</i>_dim value is strongly affected by the size of the <i>C</i>-terminal end group. The tripeptides <b>2</b> are also excellent organogelators of aromatic solvents

MOD can be crosslinked to rat liver ribosome and P2.

<p>Crosslinking reactions were carried with individual proteins (lanes 1 and 3, 4 and 6) or mixtures of two proteins (lanes 2 and 5) and subject for western analysis. Protein bands were detected by anti-myc, anti-MOD and anti-P antibodies.</p

Relative <i>N</i>-glycosidase activities of maize RIP and its variants on 28S rRNA and rat liver ribosome.

<p>Relative <i>N</i>-glycosidase activities of maize RIP and its variants on 28S rRNA and rat liver ribosome.</p

Lys158–Lys161 on MOD are responsible for the interaction between maize RIP and P2.

<p><b>A) MOD does not bind to P2 when Lys158–Lys161 are mutated to alanine.</b> SDS-PAGE of the last wash (W) and elution (E) obtained from the pull-down assay of maize RIP and P2. Input indicates the purified proteins loaded to the column. <b>B) Sensorgram showing MOD but not pro-RIP and MOD [K158A-K161A] interact with sensor chip-immobilized P2.</b> 500 nM of maize RIP variants or running buffer were injected to the sensor chip for three minutes for association, followed by a dissociation using running buffer. Response unit (RU) was monitored in a real-time manner.</p

Interaction between RIPs and P2.

<p><b>A) Interaction of MOD, TCS and StxA with P2 at different ionic strengths.</b> Pull-down assay was conducted on RIPs under various ionic strengths to compare their strength of interaction with P2. Input indicates the same amount of purified RIPs was loaded to the P2-sepharose column and proteins were eluted using buffer with 1M NaCl. <b>B) Interactions between RIPs and C-terminal truncated P2.</b> C-terminal truncated P2 variants were subject to pull down assay with RIPs. The C-terminal amino acid sequences of P2, P2 [ΔC5] and P2 [ΔC10] are: AEEKKDEKKEESEE<b>SDDDMGFGLFD</b>, AEEKKDEKKEESEE<b>SDDDMG</b> and AEEKKDEKKEESEE<b>S</b> respectively. The bold letters refer to the conserved residues in P-proteins.</p

Residues on MOD that correspond to the C11-P interacting residues on TCS.

<p><b>A) Superimposed structures of MOD (pdb: 2PQI) and TCS-C11-P complex (pdb: 2JDL).</b> The beta strands β7 and β8 in TCS (wheat) are replaced by the helix αI in MOD (cyan) while helices αG and αJ are conserved. P2-binding residues on MOD and TCS are distant apart as indicated in red and green respectively. <b>B) Comparison of C11-P interacting residues on TCS and the corresponding residues on MOD.</b> Structures of MOD and TCS are superimposed to locate the residues on MOD (colored in cyan and labelled in black and italic) corresponding to the C11-P interacting residues on TCS (colored in wheat and labelled in red). Many residues in these two RIPs are different, especially those at the hydrophobic patch of TCS (Phe-166, Val-223, Ile-225, Gly-231, Val-232 and Asn-236), suggesting MOD may interact with C11-P at a different site. <b>C) Stereo image zooming in the hydrophobic pocket of MOD (colored in cyan) with C11-P (colored in yellow).</b> The model reveals Arg-275 on MOD may crash with Leu-9 on C11-P while Glu-272 confronts directly Asp-11. Residues on TCS that interact with C11-P are highlighted in wheat for reference.</p

Screening of basic residues on MOD that are responsible for ribosome binding.

<p>The indicated residues were mutated to alanine and screened for their abilities to bind ribosome. W and E denote last wash and elution respectively.</p

Pull-down assay on TCS variants suggests that K173, R174 and K177 are involved in binding P2

<p><b>Copyright information:</b></p><p>Taken from "Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses"</p><p></p><p>Nucleic Acids Research 2007;35(5):1660-1672.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865052.</p><p>© 2007 The Author(s).</p> TCS () or its variants (–) were loaded to a P2-coupled NHS-Sepharose pre-equilibrated with binding buffer. Bound protein was eluted with 1 M NaCl in 20 mM Tris/HCl buffer pH 8.0. Fractions containing unbound protein collected during washing (W) and bound protein collected during elution (E) were analysed in 15% SDS-PAGE stained with Coomassie blue. As indicated by the presence of TCS in the wash fraction, substitution of alanine at K173, R174 and K177 positions decreases the binding of TCS on P2-coupled column (b–d). Triple-alanine substitutions in these residue positions resulted in a TCS variant (K173A/R174A/K177A) that was unable to bind P2 ()

Interaction between TCS and ribosome was compromised by K173A/R174A/K177A triple-alanine substitutions

<p><b>Copyright information:</b></p><p>Taken from "Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses"</p><p></p><p>Nucleic Acids Research 2007;35(5):1660-1672.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865052.</p><p>© 2007 The Author(s).</p> () . Rat ribosome was loaded to NHS-Sepharose coupled with TCS or its triple-alanine (K173A/R174A/K177A) variants. After extensive washing, the bound proteins were eluted with 1 M NaCl, and detected by western blot using anti-P antibody. Ribosomal proteins P0, P1 and P2 were pull-down by wild-type TCS (lane 2), while the interaction between ribosome and the triple-alanine variants (lane 1) was greatly reduced to that similar to the control (lane 3), in which the faint band of P0 was due to non-specific interactions between ribosome and the uncoupled resins. () . After rat ribosome was incubated with TCS or the triple-alanine variants in room temperature for 20 min, DSS was added to induce cross-linking between TCS and ribosomal proteins, and cross-linking product was detected by western blot using anti-P or anti-TCS antibodies. A protein band at ∼66 kDa, corresponding to the size of TCS–P0 complex, was detected by both anti-P and anti-TCS antibodies when ribosome was cross-linked with wild-type TCS (lane 2), but not with the triple-alanine variants (lane 5) and in other negative controls (lanes 1 and 4: without addition of ribosome; lanes 3, 6 and 8: without addition of DSS; lanes 7 and 8: without addition of TCS or its variants)