Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 2. Intracellular analysis of repressor pairs simulating Hdm2–p53 and Hdmx–p53 interactions in the E. coli reporter strain. (A and B) Growth analysis through droplet inoculation of serially diluted strains (10–106 colony forming units (cfu)) expressing the Hdmx–p53 pair of DBD fusions (row 1), the Hmd2–p53 pair of DBD fusions (row 2), a constitutively active repressor control (row 3) (Ref. 22) and an unrepressed (DBDs only) control (row 4) on a (A) non-selective inducing medium (LB with 100 lM IPTG) and (B) selective inducing medium (LB with 100 lM IPTG and 25 lg/mL kanamycin). (C) Reporter-gene (b-galactosidase) activity analysis of Hdm2–p53 and Hdmx–p53 strains. The arrow indicates the expression level (32 lM) for achieving approximately 90% reduction in the reporter expression to be used in the selection procedure.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Expressing, Control, Activity Assay, Selection

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 3. SDS–PAGE analysis of the affinity capture–elution assay performed with the selected SICLOPPS hits. The unspliced constructs containing sequences CIFYYV and CDLRWF were immobilized via chitin-binding domain (CBD) fusion fragments on chitin beads and incubated with an equimolar mixture of Hdm2 or Hdmx (1 mM). Retained materials were subsequently treated with a solution (1 mM) of a p53-derived peptide (ETFSDLWKLL), and the eluate as well as other components of the assay were analyzed by SDS–PAGE. The lane assignments are as follows: lane 1 is a protein ladder; lanes 2 and 3 correspond to the CIFYYV and CDLRWF SICLOPPS constructs, respectively, isolated by chitin beads from crude overexpression lysates; lanes 4 and 5 contain purified Hdm2 and Hdmx, respectively; lane 6 corresponds to an equimolar mixture of Hdm2 and Hdmx; lanes 7 and 8 contain protein material eluted with the p53-derived peptide from the affinity supports containing CIFYYV and CDLRWF leads, respectively, which were pretreated with the equimolar mixture of Hdm2 and Hdm; lanes 9 and 10 were loaded with the post-elution material retained by the chitin beads pre-functionalized with the CIFYYV and CDLRWF constructs, respectively.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: SDS Page, Construct, Binding Assay, Incubation, Derivative Assay, Isolation, Over Expression

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 4. Performance of the selected SICLOPPS constructs and the corresponding single-alanine mutants in the reporter-gene and growth rate assays. (A and B) ONPG assay data and droplet inoculation analysis, respectively, of the anti-Hdm2 CIFYYV construct and its mutants in the Hdm2–p53 strain. (C and D) ONPG assay data and droplet inoculation analysis, respectively, of CDLRWF and its mutants in the Hdmx–p53 strain.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Construct

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 5. Surface representations of Hdm2 (PDB: 1YCR; left) and Hdmx (PDB: 3DAB; right) in bound states with ligands (p53 residues 15–29) not shown for clarity. The proteins are colored according to the elemental make-up (C, gray; O, red; N, blue; S, yellow). The p53-binding pockets with labeled Leu, Trp and Phe subsites (green) are outlined to highlight the topological differences in the respective binding pockets. The residues proposed to be responsible for differences in ligand recognition patterns (F86 and H96 in Hdm2; L85 and P95 in Hdmx) are indicated by arrows.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Binding Assay, Labeling