PPAR Isotype Selective Ligands

Peroxisome proliferator-activated receptors (PPARs), are members of the steroid retinoid nuclear receptor family of ligand-activated transcription factors. There are three isotypes of the PPAR family, PPAR-a, PPAR-J and PPAR-y. Various natural fatty acids (FAs) and eicosanoids serve as ligands for the PPARs. Interestingly, several unsaturated FAs that activate the PPARs in vitro have pharmacological effects similar to those reported for the synthetic PPAR ligands. Based upon these observations...

Expression of Wild Type Ecotin

The following protocol for the expression and purification of WT eco and many of its variants is a compilation of current strategies from the Craik, Fletterick and La zarus groups 4, 11, 12 . Eco is expressed in the bacterial strains JM101 or BL21, or an E. coli ecotin knockout strain, IMAecoJ, generated in the Craik Lab (Ian Murray, unpublished results). Eco variants are generated in the pBS vector using the Kunkle mutagenesis protocol 13 , cut with BamHI and HindIII (New England BioLabs), and...

Microfluidics Method and Design

In the same way that miniaturization has impacted the electronics industry, mi-crofluidic technologies promise to spark a revolution in the biological sciences by integrating ultra small-volume sample processing within a chip format. The use of nanoliter reaction volumes and highly scaleable parallel sample processing make microfluidic technologies ideally suited to protein crystallography, where the screening and processing of precious reagents is required. Beyond reduction in sample...

Selectivity of RAR Ligands and RAR Isotypes

A very elegant experiment has been reported where enantiomers of an unnatural RAR-y-selective ligand have been determined 28 . Of special significance is the observation that one of the enantiomers is effectively inactive in the biochemical assays. While acquiring co-crystal structures of many active ligands with the same protein is now commonplace, obtaining structures of inactive (i.e., weakly active) compounds is more challenging. This work provides a high-resolution structure (PDB entry...

Tertiary Structure

A great level of structural knowledge has accumulated for protein kinases over the past decade (for various reviews see 2-9 ). The minimal kinase domain consists of approximately 300 amino acids for example, Cyclin-Dependent Kinase 2 (CDK2) has 298 residues (Fig. 2.1) 10, 11 . The kinase domain can be further divided into two lobes or sub-domains, N-terminal and C-terminal, with the lobes connected via a five to six residue hinge or linker region. The N-terminal lobe is the smaller subunit and...

What is Ecotin

Ecotin (eco) is a potent inhibitor of serine proteases that is derived from Escherichia coli. It was originally named for its ability to inhibit trypsin (E. coli trypsin inhibitor), but it is known to interact with and inhibit virtually all characterized tryp-sin-fold serine proteases. It is insensitive to the active site P1 preference of the protease (the amino acid N-terminal to the cleaved or scissile bond1)) and inhibits proteases with specificity towards basic, large hydrophobic, small...

Removing Protein Heterogeneity by Point Mutation

It is well known that post-translational modifications are another source of heterogeneity for proteins. There are at least 150 different kinds of covalent modifications of proteins, such as glycosylation, phosphorylation, S-thiolation, etc. 15 . Although the modification itself may be very small, like the addition of a single phosphate group, it may serve a physiological role and cause substantial conformational changes in the protein. Post-translational modification of a protein usually...

Confirmation of the Binding Mode

In order to investigate the binding mode of6 more comprehensively, we performed a docking study between the Cdk4 model and 6. As a result, four patterns of binding modes were suggested 18 . Considering the structural requirements for Cdk2 4 inhibitors Fig. 6.2 and the SAR of the informer libraries, we selected the binding mode candidate shown in Fig. 6.9 B. In this binding mode, there is some degree of steric repulsion between the pyridine ring and the terminal benzene ring in the fluorenone....

Other Antibiotics that Bind to the 50S Subunit 4391 Sparsomycin

Sparsomycin inhibits protein synthesis in all organisms and consequently is not used as an antibiotic. However, it has been tested extensively for its antitumor activity 49 . Sparsomycin contains a pseudouracil base with a conjugated link to a sulfur containing tail Fig. 4.11 . This chemical structure has been compared with that of puromycin, and it has been suggested that sparsomycin binds to the A-site much like puromycin 50 . Sparsomycin does not bind to the ribosome unless a P-site...

Crystallization and Analysis of Serine Proteases with Ecotin 171

Fletterick 7.2.1 Expression of Wild Type Ecotin 173 7.2.2 Purification of Wild Type Ecotin 174 7.2.3 Crystallization of Ecotin and Protease Complexes 175 7.3 Representative Examples of Ecotin and Protease Structures 175 7.3.1 Ecotin Defines the S7 Through S4' Subsites of Collagenase 176 Ecotin as a Tight Binding Substrate 177 Ecotin Defines Regions Distal to the Factor Xa Protease Domain 178 Crystallization and Structure Determination of E2P2 Complexes 180...

Crystallization and Analysis of Serine Proteases with Ecotin

Fletterick Abstract Ecotin is a macromolecular inhibitor of serine proteases. It is remarkable in binding and blocking activity of virtually all serine proteases with the canonical Asp-His-Ser catalytic triad, regardless of the amino acid sequence or substrate specificity. It forms a heterotetramer that inhibits the protease by presenting a substratelike, 11 amino acid loop to the active site. Since ecotin mimics a substrate, it defines the amino acids of the...

Nuclear Hormone Receptors Ligand Binding Domains

Nuclear hormone receptors NHR are multidomain proteins that function as transcription factors. They contain a central DNA binding domain DBD responsible for targeting the receptor to highly specific DNA sequences comprising a response element. The DBD is surrounded by two activation domains the activation function 1 AF-1 domain that resides at the N-terminus and the activation function 2 AF-2 domain that resides at the C-terminal ligand-binding domain LBD 1, 2 . NHRs for which no natural ligand...

Chemical Inducers of Dimerization to Control Transcription

Rapamycin Analogs

Chemical inducers of dimerization CID are bivalent small molecules that bind two proteins simultaneously. The purpose of these molecules is to bring the proteins together to induce signal transduction 27 . For brevity, we will limit our discussion to CIDs that directly control transcription. The basic architecture of these systems consists of two chimeric proteins. The first contains a DNA-binding domain DBD fused to a ligand-binding domain LBD and the second chimera contains an LBD and an...