Graham J. Cotton

Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation.

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.

Introduction of unnatural amino acids into proteins using expressed protein ligation.

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.

PEPTIDE LIGATION AND ITS APPLICATION TO PROTEIN ENGINEERING

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.

Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-Crk II

The Crk II adaptor protein encodes an SH2/SH3-domain containing adaptor protein with an SH2–SH3–SH3 domain structure that transmits signals from tyrosine kinases. The two SH3 domains are separated by a 54 amino acid linker region, whose length is highly conserved in xenopus, chicken, and mamalian Crk II proteins. To gain a better understanding into the role of the C-terminal region of Crk, we generated a series of C-terminal SH3 domain and SH3 linker mutants and examined their role in tyrosine kinase pathways. Expression of point mutations in the C-terminal SH3 domain (W276K Crk), at the tyrosine phosphorylation site (Y222F Crk II), or truncation of the entire C-terminus (Crk I or Crk Δ242), all increased c-Abl binding to the N-terminal SH3 domain of Crk and, where relevant, increased Tyr222 phosphorylation. Deletion analysis of c-Crk II also revealed the presence of a C-terminal segment important for trans-activation of FAK. Such mutants, Crk Δ255 or Crk Δ242 Extended Linker (Crk Δ242[EL]), characterized by a disruption in the SH3 linker/C-terminal SH3 boundary, induced robust hyperphosphorylation of focal adhesion kinase (FAK) on Tyr397, hyperphosphorylation of focal adhesion proteins p130cas and paxillin and increased focal adhesion formation in NIH3T3 cells. The effects of Crk Δ242[EL] could be abrogated by co-expression of dominant negative c-Src or the protein tyrosine phosphatase PTP–PEST, but not by dominant negative Abl. Our results suggest that the C-terminal region of Crk contains negative regulatory elements important for both Abl and FAK dependent signal pathways, and offers a paradigm for an autoinhibitory region in the SH3 linker/C-terminal SH3 domain.

Fluorescent monitoring of kinase activity in real time: development of a robust fluorescence-based assay for Abl tyrosine kinase activity

Fluorescent biosensors hold great promise for drug discovery. Using a solid-phase version of protein semi-synthesis, we incorporated two fluorophores at specific sites within a truncated version of the c-Crk-II protein. The resulting fluorescent protein biosensor permits the real-time monitoring of Abl kinase activity and provides a robust and rapid method for assaying Abl kinase inhibitors.

Synthesis of Multi-Domain Proteins Using Expressed Protein Ligation: Strategies for Segmental Isotopic Labeling of Internal Regions

Abstract Here we describe how a sequential version of the protein semi-synthesis technique, Expressed Protein Ligation (EPL), can be used to assemble multiple (i.e. 3 or more) recombinantly-derived polypeptides segments into a target protein. Sequential EPL was successfully used to assembly the 304 amino acid eukaryotic adaptor protein, Crk-II, from three recombinant polypeptide segments in good yield. Moreover, the resulting multi-component ligation product was found to possess the expected biological activity in a series of ligand binding studies. By allowing the controlled assembly of 3 or more recombinant polypeptide segments, sequential EPL opens the door to the segmental isotopic labeling of internal regions of large proteins with NMR probe-nuclei.

Expressed protein ligation: A new tool for studying protein structure and function

We are interested in using chemistry-driven protein engineering approaches to study the structure and function of the protein tyrosine kinase, c-Abl, oncogenic forms of which are implicated in the pathogenesis of virtually all chronic myelogenous leukemias, as well as some acute lymphocytic leukemias. Recently, we introduced a biosynthetic technology, Expressed Protein Ligation (EPL), which allows unnatural amino acids and biochemical/biophysical probes to be site-specifically incorporated into large proteins such as Abl and its substrates [1]. Using this protein semi-synthesis approach, we have incorporated fluorescent probes at key positions in both the c-Abl regulatory apparatus and the adapter protein c-Crk (which is phosphorylated on a single tyrosine by c-Abl). Preliminary studies indicate that these protein biosensors are useful tools for studying Abl structure and function [2].

Fluorescent Biosensor for CrkII Phosphorylation by the Abl Tyrosine Kinase

Chronic myeloid leukemia (CML) causes the excessive proliferation of myeloid cells ADDIN ENRfu [1]. The majority of CML cases are caused by the reciprocal chromosomal translocation of the first exon of c-Abl on chromosome 9 and the breakpoint cluster region (Bcr) of chromosome 22. The c-Abl gene encodes a nonreceptor kinase that catalyzes the transfer of phosphate from ATP to tyrosine residues on protein substrates. The Bcr-Abl fusion protein produces an overactive kinase, and this deregulated tyrosine kinase activity is required for leukemic transformation.