Search

We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cells control their own death through a program termed apoptosis, which is indispensable for development and homeostasis in all metazoans. Lysosomal cysteine proteases are not normally thought of as participating in apoptosis; however, recent reports have shown that the cathepsin proteases can be released from the lysosome during apoptosis, where they can participate in cell death. We report here the development of an activity-based probe that, under optimized conditions, reports on cathepsin B activity only in apoptotic cells by reading out the release of cathepsin B from the lysosomes. Biochemical characterization of apoptosis in cells from cathepsin B null mice shows delayed and suboptimal activation of caspases. Our data further supports a role for cathepsin B in the cytosol as a positive regulator of a cell death feed-forward loop and provides a chemical tool for future investigations.

The sigma factors are the key regulators of bacterial transcription initiation. Through direct read-out of promoter DNA sequence, they recruit the core RNA polymerase to sites of initiation, thereby dictating the RNA polymerase promoter-specificity. The group 1 sigma factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are autoregulated by an N-terminal sequence known as sigma1.1. We report the solution structure of Thermotoga maritima sigmaA sigma1.1. We additionally demonstrate by using chemical crosslinking strategies that sigma1.1 is in close proximity to the promoter recognition domains of sigmaA. We therefore propose that sigma1.1 autoinhibits promoter DNA binding of free sigmaA by stabilizing a compact organization of the sigma factor domains that is unable to bind DNA.

Generations of chemists and biologists have conducted research on natural products and other metabolites produced by bacteria and other microorganisms. This has led to an explosion in knowledge concerning the mechanism by which such natural products are made, ultimately allowing custom redesign of many of these molecules for increased potency and selectivity as therapeutic drugs. Along the way, scientists have begun to appreciate that the bacterial world is teeming with life on a scale hardly conceivable, with constant communication within the bacterial world and with outside neighbors, such as plants and mammals. Only in recent years have some of the signaling molecules that comprise these elaborate forms of communication been characterized in any sort of chemical detail, which has in turn peaked interest in the intricate biology of this micro-world and its interactions with the macro-world.

Background: The site-specific chemical modification of proteins has proved to be extremely powerful for generating tools for the investigation of biological processes. Although a few elegant methods exist for engineering a recombinant protein at a unique position, these techniques cannot be easily extended to allow several different chemical probes to be specifically introduced into a target sequence. As such multiply labeled proteins could be used to study many biological processes, and in particular biomolecular interactions, we decided to investigate whether such protein reagents could be generated using an extension of the semisynthesis technique known as expressed protein ligation., Results: A solid-phase expressed protein ligation (SPPL) technology is described that enables large semisynthetic proteins to be assembled on a solid support by the controlled sequential ligation of a series of recombinant and synthetic polypeptide building blocks. This modular approach allows multiple, different chemical modifications to be introduced site-specifically into a target protein. This process, which is analogous to solid-phase peptide synthesis, was used to dual-label the amino and carboxyl termini of the Crk-II adapter protein with the fluorescence resonance energy transfer pair tetramethylrhodamine and fluorescein, respectively. The resulting construct reports (through a fluorescence change) the phosphorylation of Crk-II by the nonreceptor protein tyrosine kinase, c-Abl, and was used to probe the protein-protein interactions that regulate this important post-translational process., Conclusions: SPPL provides a powerful method for specifically modifying proteins at multiple sites, as was demonstrated by generating a protein-based biosensor for Crk-II phosphorylation. Such protein derivatives are extremely useful for investigating protein function in vitro and potentially in vivo. This modular approach should be applicable to many different protein systems.

The ability to assemble a target protein from a series of peptide fragments, either synthetic or biosynthetic in origin, enables the covalent structure of a protein to be modified in an unprecedented fashion. The present technologies available for performing such peptide ligations are discussed, with an emphasis on how these methodologies have been utilized in protein engineering to investigate biological processes.

Background: Modifying the covalent structure of a protein is an effective empirical route to probing three-dimensional structure and biological function. Here we describe a combinatorial protein chemistry strategy for studying structure-activity relationships in proteins. Our approach (termed 'protein signature analysis') involves functional selection from an array of self-encoded protein analogs prepared by total synthesis, coupled to a simple chemical readout that unambiguously identifies the modified proteins in the resulting active and inactive populations., Results: Protein signature analysis was used to study the interaction of the amino-terminal SH3 domain from the cellular adaptor protein c-Crk with its cognate proline-rich peptide, C3G. Using a functional selection assay, the qualitative effects of scanning a series of synthetic analog units through the amino-acid sequence of the SH3 domain were evaluated. The analog units were designed to alter both amino-acid sidechains and the polypeptide backbone within the protein. These chemical studies revealed that the sidechain of Asp 150 in the SH3 domain is essential for ligand binding and that changes in the structure of the polypeptide backbone can also result in loss of binding activity., Conclusions: These chemical studies have provided new insight into how ligand binding is related to the covalent structure of the SH3 domain. Protein signature analysis is a powerful and conceptually novel way of studying the molecular and chemical basis of protein function; it combines the advantages of systematic modification of a protein's chemical structure with the practical convenience of combinatorial synthesis.