Protein semisynthesis and expressed protein ligation: chasing a protein's tail

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The adaptation of native chemical ligation to protein semisynthesis has become a powerful way to address problems in the analysis of protein structure and function. In particular, the exploitation of nature's inteins in expressed protein ligation is now a standard approach in the study of proteins. Site-specific incorporation of unnatural amino acids, biophysical probes and post-translational modifications in proteins have led to new insights into enzyme mechanisms, protein folding, ion channel function, translation and signaling.

Introduction

Providing a complete understanding of protein structure and function has emerged as one of the great challenges in biochemistry in the postgenomics era. In the analysis of proteins, the ability to manipulate the chemical structure remains an important strategy in sorting out the basis of folding, and understanding enzyme mechanisms, post-translational modification and molecular recognition. There have been several key technical advances in the past 50 years which have eased the chemical manipulation of proteins. The first generation of protein chemists used group-modifying reagents, which, in many cases, facilitate the selective tagging of proteins isolated from living organisms. Still in use today, this method is especially powerful for chemoselective targeting of cysteines with fluorescent and spin-label probes, cross-linking reagents and activity modulators. Second, and perhaps the most important step in protein analysis, has been the use of site-directed mutagenesis, which in principle enables any of the 20 encoded amino acids to be swapped, removed or inserted into protein sequences [1]. The power of this method includes its applicability for investigating proteins in vitro or in vivo. The relatively recent extension of this method to incorporate unnatural amino acids site-specifically promises to be another major advance along these lines [2]. A third crucial advance was the introduction of total chemical synthesis by ligation strategies, particularly exploiting the native chemical ligation reaction [3]. This chemical reaction enables the chemoselective linkage of an amino-terminal cysteine-containing peptide with a carboxy-terminal thioester-containing peptide. The mechanism of this reaction involves initial transthioesterification followed by S,N-shift to produce a native amide bond. The utility of this advance was expanded by its marriage to recombinant protein technology in new approaches to protein semisynthesis. This can be achieved in two ways. Using proteases, amino-terminal cysteines can be introduced into recombinant protein fragments, which can then be used for ligation [4]. Alternatively, α–thioesters can be introduced into recombinant protein fragments by fusion to inteins [5]. Inteins are modules that mediate protein splicing and are functionally analogous to RNA introns [5]. Intein-mediated thioester formation enables recombinant fragments to be used in the protein semisynthetic method known as expressed protein ligation (EPL) [6, 7]. EPL (Figure 1) has become an especially popular method for carboxy-terminal modification of proteins, in part because peptide thioester chemical synthesis is avoided. In this paper, we discuss the exploitation of native chemical ligation in protein semisynthesis in a variety of contexts. Because this area has been reviewed previously [8, 9], we focus on several recent advances which illustrate the versatility of this modern semisynthetic approach to understanding proteins.

Section snippets

Analysis of protein mechanisms with unnatural amino acids

One general area that has benefited significantly from protein semisynthesis has been analysis of protein structure and function with unnatural amino acids. Most of these recent studies have exploited EPL in which the unnatural residues (Figure 2) have been incorporated into an N-Cys-containing synthetic peptide which is then ligated to a recombinant protein thioester fragment. These studies have examined a diverse set of proteins, including Src, ribonucleotide reductase, ribonuclease, a zinc

Csk-catalyzed phosphorylation of Src

Src is a protein tyrosine kinase which is itself a substrate of Csk-catalyzed tail tyrosine phosphorylation, inhibiting the oncogenic potential of Src. The nature of the tyrosine kinase mechanism of Csk has been the subject of study for several years, and most work had been carried out with artificial peptide substrates rather than its natural protein substrate Src [10]. Because the tail tyrosine of Src, which undergoes phosphorylation, is within 10 amino acids of the carboxy-terminus, it is

Proton and electron transfer in ribonucleotide reductase

Ribonucleotide reductase (RNR) enzymes catalyze the fascinating and chemically challenging conversion of nucleotides to deoxynucleotides. They have a major role in maintaining a normal balance of DNA precursors and are the target of at least two chemotherapeutic drugs used in humans. The class I RNR enzymes utilize a tyrosyl radical cofactor which is remote from the active site Cys, separated by 35 Å [14]. Proton-couple electron transfer from the tyrosyl radical had been postulated to be a

Folding of ribonuclease A

Elucidating the protein-folding mechanism of ribonuclease A has been of intensive interest since the pioneering studies of Anfinsen. One aspect thought to contribute to folding efficiency is the stereochemistry of peptide bonds, particularly prolines. It was hypothesized that Pro-114 in ribonuclease A might enhance conformational stability and/or folding efficiency if this residue were constrained exclusively in its cis conformation [17]. To investigate this, the unnatural amino acid

DNA recognition by a zinc finger

Site-specific recognition of DNA sequences by zinc finger transcription factors is one of the fundamental mechanisms used in the regulation of gene expression. Much effort has been expended to understand the rules governing molecular recognition of specific DNA–protein contacts with the hope of engineering designer transcription factors. It was recently hypothesized that three tandem Cys2His2 zinc fingers could show altered specificity if a key Arg residue was replaced with an unnatural

Potassium channel ion function

The recent elucidation of the crystal structures of the potassium channel KcsA has led to unprecedented insights into the basis of ion selectivity versus sodium and other cations. The selectivity filter of KcsA includes Gly-77, which upon mutation to Ala results in functional loss [19]. Gly-77 exists in a left-handed helical conformation. However, its precise contribution to potassium channel function had been unclear. It was considered that a d-amino acid at this position would maintain the

Application of biophysical probes in protein analysis

Perhaps of all of the potential chemical modifications of a protein, fluorescent labeling has been the most widely used in biological research. Of the many approaches available to label a protein fluorescently, EPL offers several distinct advantages. First, the fluorophore addition is highly flexible, needing only to be linked to a cysteine moiety to facilitate the native chemical ligation reaction. Second, the stoichiometry of fluorescent labeling is usually very high. Third, fluorophore

FRET analysis of serotonin N-acetyltransferase

Serotonin N-acetyltransferase is the crucial enzyme governing melatonin regulation. In addition to catalyzing acetyl transfer from acetyl-CoA, this enzyme exhibits a promiscuous alkyltransferase activity in a slightly altered conformation of its active site [21]. Although probably not physiologically important, this alkyltransferase activity can be exploited pharmacologically to generate inhibitors in vivo. It had previously been proposed that these two activities were catalyzed in the active

Fluorescent analysis of translation initiation

Protein synthesis involves coordinated action of the ribosome, along with several protein-elongation initiation factors (eIFs). Two of these factors, eIF1A and eIF1, are required for the formation of the 43S mRNA–ribsomal subunit complex. How these initiation factors interact with the ribosome is poorly understood. Recently, both eIF1 and eIF1A were labeled at the carboxy-terminus with rhodamine by employing EPL [22]. Using fluorescence anisotropy, it was demonstrated that labeled eIF1 and

NMR analysis of selectively isotopically labeled proteins

Protein semisynthesis offers unusual opportunities in NMR-based structural analysis of domain interactions and subtle features of amino acid residue conformatisons. The ability covalently to link isotopically labeled residues with protein fragments containing natural abundance amino acids enables high signal to noise within regions of interest. A good example of this was reported within the bacterial transcription factor sigma, in which its carboxy-terminal domain was evaluated structurally in

Post-translational modifications

In terms of biological understanding, protein semisynthesis offers a great deal to unravel the complex mechanisms and functions involved in protein post-translational modification. Perhaps most importantly, semisynthetic approaches enable stoichiometric site-specific incorporation of one or more natural and nonhydrolyzable mimics of modified amino acids, such as phospho-Ser (pSer) and phospho-Tyr (pTyr) (Figure 5). In addition, semisynthesis enables an autoprocessing enzyme to be made readily

Protein phosphorylation

Reversible protein phosphorylation is a major mechanism of cell signal transduction and has been one of the most intensively studied areas in biochemistry. Phosphorylation by protein kinases and dephosphorylation by protein phosphatases are key regulators of biological function. Protein semisynthetic approaches have been applied to transforming growth factor-β signaling, in which the transforming growth factor-β receptor serine–threonine kinase (TßRI kinase) and its substrate effector SMAD2

Protein acetylation and chromatin modifications

Reversible histone and transcription factor acetylation is a major mechanism for the regulation of gene expression. The histone acetyltransferase (HAT) p300 is a key transcriptional coactivator in gene regulation but has proved challenging to characterize biochemically. The catalytic domain of this enzyme has been difficult to express in Escherichia coli, presumably because of toxicity due to hyperacetylation of host proteins. More troublesome is the fact that the recombinant HAT domain

Protein prenylation

Protein prenylation on cysteine residues is an important mode of cellular regulation of a variety of proteins, especially small G proteins (GTPases). The Ypt family of small GTPases has numerous roles in intracellular trafficking, and these GTPases are modified on their carboxy-termini by geranylgeranylation. How this prenylation governs protein–protein interactions has been unclear, and sample preparations of the prenyl-modified Ypt1 has been a technical challenge. To overcome this, EPL was

Lantibiotic biosynthetic mechanisms

The lantibiotics comprise a group of peptide-based antibiotics which are biosynthesized ribosomally and then subjected to complex post-translational modifications which are crucial for their function. These modifications include dehydration of Ser and Thr groups to dehydroalanine residues and regioselective cyclization reactions involving Cys amino acids to the unsaturated residues. Although the lantibiotics have been known about for many years, it has only recently been possible to

Conclusions and outlook

The field of protein semisynthesis has been dramatically enhanced by the integration of inteins and the native chemical ligation reaction. As illustrated in this review, precise alteration of complex proteins with unnatural amino acids, biophysical probes and post-translational modifications can be used to address a wide range of biological problems. In spite of this progress, there are several remaining challenges which limit the broader impact of this technology. Protein semisynthesis using

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank the NIH (P.A.C.) and DFG (D.S.), who support our efforts in this field. We are grateful to past and current colleagues in our laboratory and to collaborators whose ideas and experimental skills have contributed a great deal to this subject. We apologize to the authors of the many recent interesting reports in protein semisynthesis that could not be covered in this article because of space limitations.

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