Browsing by Subject "Peptide synthesis"

Now showing 1 - 4 of 4

Design, synthesis, and evaluation of conformationally-constrained Grb2 SH2 ligands and a concise total synthesis of lycopladine A
(2010-05) Delorbe, Johnathan E.; Martin, Stephen F.; Anslyn, Eric V.; Brodbelt, Jennifer S.; Liu, Hung-Wen; Siegel, Dionicio R.
Conformationally constrained ligands and their flexible analogues were prepared as inhibitors of the Grb2 SH2 domain in order to study the structural and energetic effects of ligand preorganization in protein-ligand interactions. The compounds were prepared by using trans-cyclopropane-containing amino acid mimics, macrocyclization, or [alpha,alpha]-disubstituted amino acid residues. All trans-cyclopropane containing peptides were more potent than their corresponding succinate containing analogues due to an enthalpic advantage. Surprisingly, the binding of constrained peptides to the domain was entropically disfavored relative to their flexible controls. Effects of proton transfer and desolvation as being the source of the unprecedented entropic penalty for the constrained ligands relative to their respective controls were precluded, and X-ray crystallographic studies revealed that the binding conformations for the respective cyclopropane and succinate containing ligands were similar. This led us to believe that differential changes in protein dynamics may occur upon binding of the constrained and flexible ligands, which could contribute to the observed binding energetics. Two 23-membered macrocyclic ligands were slightly more potent than their corresponding linear controls. The amino acids used to link the N- and C-termini of the linear peptides to form the macrocycles were found to affect the energetics of binding. In one case, the 23-membered macrocycle was more potent than its control due to an entropic advantage, whereas the other 23-membered macrocycle was more potent than its control because it benefited from an enthalpic advantage. [alpha,alpha]-Disubstituted and [alpha]-monosubstituted residues that varied in hydrophobic character were incorporated into Grb2 SH2 domain binding tripeptides, and binding became more favorable as nonpolar surface area increased only for the set of tripeptides possessing cyclic [alpha,alpha]-disubstituted residues. The increase in affinity was due to an increasing enthalplic term, whereas the entropy of binding became less favorable. A total synthesis of (±)-lycopladine A was achieved in five steps from known compounds. The tricyclic core of the natural product was prepared utilizing a novel two-step sequence comprising a conjugate addition of a metalated picoline derivative followed by an intramolecular enolate arylation. It was demonstrated that the natural product existed in a solvent dependent equilibrium with its isomeric lactol.
Flanking Residues Are Central to DO11.10 T Cell Hybridoma Stimulation by Ovalbumin 323–339
(Public Library of Science, 2012-10-23) Roy, Benjamin M.; Zhukov, Dmitriy V.; Maynard, Jennifer A.
T cell activation requires formation of a tri-molecular interaction between a major histocompatibility complex (MHC), peptide, and T cell receptor. In a common model system, the ovalbumin epitope 323–339 binds the murine class II MHC, I-Ad, in at least three distinct registers. The DO11.10 T cell recognizes the least stable of these, as determined by peptide-MHC dissociation rates. Using exogenous peptides and peptide insertions into a carrier protein in combination with IL-2 secretion assays, we show that the alternate registers do not competitively inhibit display of the active register four. In contrast, this weakly binding register is stabilized by the presence of N-terminal flanking residues active in MHC binding. The DO11.10 hybridoma is sensitive to the presence of specific wild-type residues extending to at least the P-3 peptide position. Transfer of the P-4 to P-2 flanking residues to a hen egg lysozyme epitope also presented by I-Ad increases the activity of that epitope substantially. These results illustrate the inherent complexity in delineating the interaction of multiple registers based on traditional thermodynamic measurements and demonstrate the potential of flanking residue modification for increasing the activity of weakly bound epitopes. The latter technique represents an alternative to substitution of anchor residues within a weakly bound register, which we show can significantly decrease the activity of the epitope to a responding T cell.
A Model of a MAPK•Substrate Complex in an Active Conformation: A Computational and Experimental Approach
(Public Library of Science, 2011-04-11) Lee, Sunbae; Warthaka, Mangalika; Yan, Chunli; Kaoud, Tamer S.; Piserchio, Andrea; Ghose, Ranajeet; Ren, Pengyu; Dalby, Kevin N.
The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)2-3-X2-6-ΦA-X-ΦB, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus 7RK-X2-ΦA-X-ΦB13. This results in a 2-fold increase in kcat/Kmfor the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves kcat/Km by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 ofEts results in a 14-fold decrease in kcat/Km, with little apparent change in kcat. A peptide that binds to the DRS of ERK2 affects Km, but not kcat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.
Thermodynamic evaluation of torsional strain in peptide backbones : studies on constrained tripeptide Grb2 adaptor protein inhibitors
(2017-08-08) White, Zachary Ryan; Martin, Stephen F.
A substantial challenge in ligand design for protein-ligand interactions is the accurate prediction of changes in binding energy associated with small structural variations in ligands. In a previous study analyzing the binding energetics of varied hydrophobic surface area in peptide mimics, we observed an unfavorable gauche conformation in the pY+3 sidechain of an inhibitor of growth receptor bound protein 2 (Grb2) SH2 domain. To investigate the change in binding energy from alleviating of torsional strain in the protein-ligand complex, constrained ligand analogues containing a cyclopropane ring were designed to reduce the energetic penalty associated with adopting a gauche conformation, potentially enhancing binding enthalpy. Additionally, the cyclopropane ring serves to preorganize the ligand by positioning the terminal substituent in a similar fashion to the bound state of the flexible control, potentially enhancing binding entropy. Thus, the proposed constrained ligands present a unique opportunity to enhance both binding enthalpy and entropy with a single modification. The synthesis of three constrained peptide mimics will be discussed. Additionally, optimization of the growth and expression of Grb2 SH2 for binding analysis with the prepared ligands will be discussed.