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Thomas Kodadek

Publications and source records attributed to Thomas Kodadek.

At least 37 records · Page 2Linked to original sources
PubMed2004

Periodate-triggered cross-linking of DOPA-containing peptide-protein complexes.

Chemical cross-linking is a powerful methodology for analyzing proteins-small molecule and protein-protein interactions. We describe the development of a new chemical cross-linking reaction for the study of protein complexes. Specifically, we show that molecules containing an ortho dihydroxyarene unit can be oxidized selectively with sodium periodate in the presence of native proteins, producing an ortho quinone intermediate that can cross-link with suitable nearby protein residues. We demonstrate the efficacy and specificity of this chemistry for a peptide-protein complex and also deduce the binding site of an artificial activation domain on a proteasome subcomplex.

Amino Acid Sequence↗
PubMed2004

Photolithographic synthesis of peptoids.

We describe a novel photolithographic approach to the synthesis of peptoids (oligo-N-substituted glycines). This strategy enables the construction of a spatially addressable peptoid microarray, thus providing a potentially powerful tool for the discovery of protein ligands.

Journal Article↗
PubMed2004

Physical and functional association of RNA polymerase II and the proteasome.

Recent studies from a number of laboratories have revealed a surprising number of connections between RNA polymerase II transcription and the ubiquitin/proteasome pathway. We now find yet another intersection of these pathways by showing that the 26S proteasome associates with regions of the GAL1, GAL10, and HSP82 genes, including the 3' ends, in a transcription-dependent fashion. The appearance of the proteasome on these inducible genes correlates with both the accumulation of transcripts and the buildup of RNA polymerase II complexes in the same region. Furthermore, the 26S proteasome and RNA polymerase II coimmunoprecipitate, and inhibition of 26S proteolytic activity leads to increased read through of a transcription termination site. We suggest that the proteasome is generally recruited to the DNA at sites of stalled RNA polymerase and may act to resolve these complexes.

Cysteine Endopeptidases↗
PubMed2004

Transformation of low-affinity lead compounds into high-affinity protein capture agents.

A simple and potentially general approach to the isolation of high-affinity and -specificity protein binding synthetic molecules is presented. A modest affinity lead compound is appended to the end of each molecule in a combinatorial library of oligomeric compounds, such as peptides or peptoids. The library is then screened under conditions too demanding for the lead to support robust binding to the protein target. It was anticipated that this procedure would select for bivalent ligands in which the oligomer library provides both a second binding element as well as an appropriate linker between this element and the lead compound. We report here synthetic ligands for the Mdm2 protein and ubiquitin able to capture their target proteins from dilute solutions in the presence of a large excess of other proteins.

Amino Acid Sequence↗
PubMed2004

Peptides as modulators of enzymes and regulatory proteins.

There is currently great interest in the development of methods to modulate the function of diverse classes of target proteins with chemicals (agonists or antagonists). These would be valuable reagents for biomedical research and some might serve as potential drug leads. Traditionally, most chemicals that modulate protein function have been enzyme inhibitors isolated in functional screens specific for the enzyme of interest. However, recent efforts from many laboratories have suggested that relatively simple binding assays may provide a more convenient and general route to chemical modulators. We review here this work with a particular emphasis on peptide modulators.

CDC2-CDC28 Kinases↗
PubMed2004

Synthetic molecules as antibody replacements.

Antibodies are by far the most versatile, valuable, and widely used protein-binding agents. They are essential tools in biological research and are increasingly being developed as therapeutic reagents. However, antibodies have a number of practical limitations, and it would be desirable in many applications to replace them with simpler, more robust synthetic molecules. Unfortunately, synthetic protein-binding agents rarely exhibit the high affinity and specificity typical of a good antibody. This article reviews efforts to overcome these limitations and to develop a facile, high-throughput methodology for the isolation of synthetic protein ligands with antibody-like binding characteristics.

Antibodies, Bispecific↗
PubMed2003

Isolation of protein ligands from large peptoid libraries.

The isolation of ligands for large numbers of proteins is an important goal in proteomics. Whereas peptide libraries are rich sources of protein-binding molecules, native peptides have certain undesirable properties, such as sensitivity to proteases that make them less than ideal for some applications. We report here the construction and characterization of large, chemically diverse combinatorial libraries of peptoids (N-substituted oligoglycines). A protocol for the isolation of specific protein-binding molecules from these libraries is described. These data suggest that peptoid libraries will prove to be inexpensive and convenient sources of protein ligands.

Amines↗
PubMed2003

Using oxidative crosslinking and proximity labeling to quantitatively characterize protein-protein and protein-Peptide complexes.

The quantitative analysis of protein-protein and protein-peptide complexes is of fundamental importance in biochemistry. We report here that nickel-catalyzed proximity biotinylation and Ru(II)(bpy)(3)(2+)-mediated oxidative crosslinking can be used to measure the equilibrium dissociation constant and stoichiometry of protein complexes. Only small amounts of protein are required, neither of the binding partners must be immobilized on a surface, and no special instrumentation is necessary. This chemistry should provide a useful complement to existing methods for the analysis of protein-protein and protein-peptide interactions.

Amino Acid Sequence↗
PubMed2003

Immobilized peptides as high-affinity capture agents for self-associating proteins.

There is currently great interest in the fabrication of protein-detecting arrays comprised of large numbers of immobilized protein capture agents. While most efforts in this arena have focused on the use of biomolecules such as antibodies and nucleic acid aptamers as capture agents, synthetic species have many potential advantages. However, synthetic molecules isolated from combinatorial libraries generally do not bind target proteins with the high affinity necessary for array applications. Here, we demonstrate that simple linear peptides bind dimeric proteins tenaciously when immobilized, although they exhibit only modest affinity in solution. These data show that high-affinity bidentate capture agents for dimeric proteins can be created by simply immobilizing modest-affinity ligands on a surface at high density, bypassing the requirement for careful optimization of linker length and geometry that is normally required to create a high-affinity solution bidentate ligand.

Combinatorial Chemistry Techniques↗
PubMed2002

Microwave-assisted solid-phase synthesis of peptoids.

Microwave irradiation reduces the reaction time for the solid-phase synthesis of peptoids. Under these conditions, coupling of each residue requires only 1 min. The purity and yields of peptoids synthesized in this way are as good as or better than those achieved using standard methods. [reaction: see text]

Amino Acid Sequence↗
PubMed2002

A nonproteolytic function of the 19S regulatory subunit of the 26S proteasome is required for efficient activated transcription by human RNA polymerase II.

We recently reported that the 19S regulatory subunit of the yeast 26S proteasome stimulates transcription elongation by RNA polymerase II. However, because of basic differences between yeast and mammals in the components and cellular location of the proteasome, it is crucial to assess whether this is a general phenomenon. Here we address this question and demonstrate that (1) the nonproteolytic activity of the 19S (PA700) complex of the proteasome is required for efficient activated transcription in the mammalian in vitro system, (2) this requirement applies to both natural and artificial activators, and (3) highly purified PA700 can provide this activity. In vitro transcription assays using HeLa cell nuclear extracts reveal that antibodies against human Trip1p/Rpt6 (mammalian Sug1p), one of the six ATPases in the PA700, significantly inhibit activated transcription. Similarly, immunodepletion of the PA700 from the extract also significantly reduces activated, but not basal, transcription and add-back of the highly purified mammalian PA700 restores the activity. Finally, inhibitors of the proteasome's peptidase activities do not affect transcription although the peptidase activity is almost completely inhibited. These findings indicate that the requirement for a nonproteolytic activity of the 19S complex in transcription is general in eukaryotes.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2002

Physical association of the APIS complex and general transcription factors.

It has recently been demonstrated that a fragment of the proteasome, called the APIS complex, plays an important role in RNA polymerase II-mediated transcription. Here, it is shown that the APIS complex is physically associated with many general transcription factors, including components of yeast FACT (Cdc68/Pob3), TFIID, TFIIH, and the RNA polymerase II holoenzyme. Depletion of this APIS transcription factor complex from a yeast whole cell extract resulted in reduced transcription, indicating that it is functionally relevant. The APIS/transcription factor complex does not include detectable levels of the 20S proteolytic sub-unit of the proteasome. Furthermore, immunopurified 26S proteasome contains little or no transcription factors, suggesting that transcription factors and the 20S bind competitively to the APIS complex. These data add to the growing body of evidence that the APIS complex has a role in transcription, independent of its role in proteolysis and, furthermore, argues that it functions in association with the general transcription complex.

Adenosine Triphosphatases↗
PubMed2002

Removal of impurities from transcription factor preparations that alter their DNA-binding properties.

Biochemical studies of transcriptional activators are important for understanding their detailed mechanism of action. Such experiments generally employ chimeric constructs comprised of fused DNA- binding and activation domains that are expressed in, and purified from, Escherichia coli, since full-length activators are usually difficult to express. We report here that such preparations contain chaperone impurities that affect the DNA-binding properties of the activator, for example sharply reducing the half-life of the protein-DNA complex. A simple method to remove these troublesome contaminants is described.

Adenosine Triphosphate↗