Christopher J. Noren

Biosynthetic method for introducing unnatural amino acids site-specifically into proteins

Publisher Summary This chapter discusses a biosynthetic method for introducing unnatural amino acids site-specifically into proteins. This method involves the replacement of the codon for the amino acid of interest with the amber nonsense codon by conventional oligonucleotide-directed mutagenesis. The amber nonsense codon is not recognized by any of the common tRNAs involved in protein synthesis, and thus can be viewed as a “blank” in the genetic code. A suppressor tRNA is then constructed that recognizes the amber nonsense codon. The suppressor tRNA is then chemically aminoacylated with the desired unnatural amino acid and is added to an in vitro transcription–translation system programmed with the mutagenized DNA. This results in the specific incorporation of the unnatural amino acid at the position corresponding to the amber mutation. The chapter describes preparation of the suppressor tRNA, general methodology for chemical aminoacylation of the suppressor tRNA, and an in vitro transcription–translation system optimized for the incorporation of unnatural amino acids into proteins.

Dissecting the Chemistry of Protein Splicing and Its Applications

Protein splicing, the protein equivalent of RNA splicing, is a newly discovered posttranslational process that proceeds through a branched protein intermediate and produces two separate polypeptides from one gene. The experimental data used to distinguish among the proposed protein-splicing mechanisms are presented along with the progress made towards fully describing the mechanism. Numerous protein engineering applications using modified inteins have been developed, including the generation of alpha-thioesters in proteins, which circumvent the limits of solid-phase peptide synthesis.

Expanding the Genetic Code of Escherichia coli with Phosphoserine

Engineered bacterial translation can be used to direct site-specific insertion of an amino acid into proteins. O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNASep binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep218, Sep222). This system has general utility in protein engineering, molecular biology, and disease research.

Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP‐tag fusion proteins in living cells. SNAP‐tag is an engineered variant of the human repair protein O6‐alkylguanine‐DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP‐tag protein. We describe the use of intramolecularly quenched probes for wash‐free labeling of cell surface‐localized epidermal growth factor receptor (EGFR) fused to SNAP‐tag and for direct quantification of SNAP‐tagged β‐tubulin in cell lysates. In addition, we have characterized a fast‐labeling variant of SNAP‐tag, termed SNAPf, which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAPf and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.

Single M13 bacteriophage tethering and stretching

The ability to present biomolecules on the highly organized structure of M13 filamentous bacteriophage is a unique advantage. Where previously this viral template was shown to direct the orientation and nucleation of nanocrystals and materials, here we apply it in the context of single-molecule (SM) biophysics. Genetically engineered constructs were used to display different reactive species at each of the filament ends and along the major capsid, and the resulting hetero-functional particles were shown to consistently tether microscopic beads in solution. With this system, we report the development of a SM assay based on M13 bacteriophage. We also report the quantitative characterization of the biopolymers elasticity by using an optical trap with nanometer-scale position resolution. Expanding the fluctuating rod limit of the wormlike chain to incorporate enthalpic polymer stretching yielded a model capable of accurately capturing the full range of extensions. Fits of the force-extension measurements gave a mean persistence length of ≈1,265 nm, lending SM support for a shorter filamentous bacteriophage persistence length than previously thought. Furthermore, a predicted stretching modulus roughly two times that of dsDNA, coupled with the systems linkage versatility and load-bearing capability, makes the M13 template an attractive candidate for use in tethered bead architectures.

Site-specific mutagenesis with unnatural amino acids

The incorporation of unnatural amino acids into proteins by site-specific mutagenesis provides a valuable new methodology for the generation of novel proteins that possess unique structural and functional features.

Protein-Spleißen: Mechanismus und Anwendungen

Protein-Spleisen, das Aquivalent des RNA-Spleisens auf Proteinebene, ist ein neu entdeckter posttranslationaler Prozess, der uber ein verzweigtes Protein-Intermediat zur Bildung zweier verschiedener Polypeptide fuhrt, die sich vom selben Gen ableiten. Die experimentellen Befunde zur Aufklarung des vollstandigen Mechanismus des Protein-Spleisens sowie zahlreiche Anwendungen fur das Protein-Engineering unter Verwendung von modifizierten Inteinen werden beschrieben, darunter die Einfuhrung von α-Thioesterbindungen in Proteine, wodurch die Beschrankungen der Festphasensynthese von Peptiden umgangen werden.

The appearance of pyrrolysine in tRNAHis guanylyltransferase by neutral evolution

tRNAHis guanylyltransferase (Thg1) post-transcriptionally adds a G (position −1) to the 5′-terminus of tRNAHis. The Methanosarcina acetivorans Thg1 (MaThg1) gene contains an in-frame TAG (amber) codon. Although a UAG codon typically directs translation termination, its presence in Methanosarcina mRNA may lead to pyrrolysine (Pyl) incorporation achieved by Pyl-tRNAPyl, the product of pyrrolysyl-tRNA synthetase. Sequencing of the MaThg1 gene and transcript confirmed the amber codon. Translation of MaThg1 mRNA led to a full-length, Pyl-containing, active enzyme as determined by immunoblotting, mass spectrometry, and biochemical analysis. The nature of the inserted amino acid at the position specified by UAG is not critical, as Pyl or Trp insertion yields active MaThg1 variants in M. acetivorans and equal amounts of full-length protein. These data suggest that Pyl insertion is akin to natural suppression and unlike the active stop codon reassignment that is required for selenocysteine insertion. Only three Pyl-containing proteins have been characterized previously, a set of methylamine methyltransferases in which Pyl is assumed to have specifically evolved to be a key active-site constituent. In contrast, Pyl in MaThg1 is a dispensable residue that appears to confer no selective advantage. Phylogenetic analysis suggests that Thg1 is becoming dispensable in the archaea, and furthermore supports the hypothesis that Pyl appeared in MaThg1 as the result of neutral evolution. This indicates that even the most unusual amino acid can play an ordinary role in proteins.

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40-50-fold enhancement in affinity over methyl α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins—ConA, LcH, and PSA—that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

Enabling Glycosyltransferase Evolution: A Facile Substrate‐Attachment Strategy for Phage‐Display Enzyme Evolution

Directed enzyme evolution has the potential to generate novel catalysts able to compete with chemical methodologies in the synthesis of complex biomolecules. Directed evolution is often carried out in cells, but for many classes of enzymes, such as glycosyltransferases, it is difficult to couple enzymatic activity to a cell-based selection. M13 phage display is an in vitro methodology that potentially enables the selection of enzymes that do not provide cell-based phenotypes. In order to select phage-bound enzymes based on catalytic activity, the desired substrate needs to be immobilized near the displayed enzyme to allow for affinity capture of the desired product. Schultz and co-workers developed the first catalysis-based display method, in which the substrate molecule was attached next to the displayed enzyme by a coiled-coil interaction. Although this approach is promising, only a handful of enzymes have been displayed on phage in an active form, and there are still no facile, general methods for substrate immobilization on the phage (vide infra). Here we introduce a chemically straightforward method for substrate attachment using selenocysteine (Sec) residues. Implementation of this method required construction of a phagemid/helper phage system that allows the display of pIII fusions bearing Sec residues adjacent to a displayed enzyme. We also report the first display of an active glycosyltransferase on phage. This work lays the foundation for evolving glycosyltransferases to make novel glycoconjugates and should also enable the directed evolution of other enzyme classes for which ex vivo selection strategies are desirable. Phage display is a convenient strategy to link phenotype and genotype; it enables amplification and identification of peptides and proteins following an in vitro selection. In phage-display enzyme evolution, proximal substrate attachment can be accomplished by using an M13 phagemid/helper phage system in which two different types of pIII fusions are presented on the phage surface (see Scheme 1). The enzyme is displayed on a pIII fusion expressed from the phagemid; the adjacent pIII peptides expressed from the helper phage genome provide potential sites for substrate attachment. Widespread implementation of phage-display enzyme evolution requires a method of modifying a uniquely reactive handle with a functionalized substrate that can be accessed with minimal synthesis. One of us (C.J.N.) has shown recently that selenocysteine (Sec), which is encoded by the TGA codon, can be expressed on phage by fusing a cassette consisting of a TGA codon followed by a selenocysteine insertion sequence (SECIS) to the 5’ end of gIII. Because Sec is considerably more nucleophilic than cysteine and reacts at lower pH (pKa of 5.2 versus 8.1 for Cys), Sec-bearing phage can be derivatized selectively in the presence of other potential side-chain nucleophiles. Encouraged by these results, we wanted to evaluate selenocysteine as a handle for substrate immobilization on phage displaying a pIII-enzyme fusion. We prepared helper phage encoding pIII bearing an N-terminal Sec residue by performing a vector swap between a M13KE vector containing the SECIS insert and M13KO7 using the PacI and BsrGI sites. We also prepared a phagemid (pMurG-pIII) encoding the E. coli glycosyltransferase (Gtf) MurG, an enzyme essential in bacterial cell-wall biosynthesis. The murG gene was subACHTUNGTRENNUNGcloned from a pET-21a expression vector (Novagen) into the pFAB5cHis.TT.HUI phagemid vector as a fusion to the 3’-end of a pelB leader sequence and the 5’-end of a truncated gIII. MurG was chosen as starting point to explore enzyme evolution by phage display because it is a member of the GT-B superfamily of Gtfs that have related three dimensional structures but different substrate selectivity. Many members of the GT-B superfamily are involved in antibiotic biosynthesis, and the ability to alter the substrate selectivity of these Gtfs could enable the production of new biologically active glycoconjugates that are synthetically difficult to access. Infection of cultures containing the pMurG-pIII phagemid with the Sec-expressing helper phage resulted in the production of phage bearing both the enzyme and the Sec handle on the same end of the phage particle (Scheme 1). As a negative control for Sec modification, we also prepared phage expressing wild-type pIII by infecting bacteria containing the pMurGpIII phagemid with the commercially available M13KO7 helper phage. All phage were purified by using CsCl gradient centrifugation. The activity of phage-bound MurG was established by incubating phage with UDP-[C]-GlcNAc and a biotin-labeled lipid I analogue. Product formation was monitored by spotting the reaction mixtures onto streptavidin membranes at various time points (Scheme 2). The membranes were washed and counted to evaluate the product formation. Radioactive counts above background were detected on the membranes within a few minutes and continued to increase for approximately 1 h before leveling off (Figure 1). Using the specific activity of the radiolabeled UDP-GlcNAc (30 mCi mmol ), we estimate that 0.1 mm product was formed within an hour; this means that each phage-bound MurG performed between 10 and 10 turnovers. Only a few phage-bound enzymes reported have been [a] Dr. K. R. Love, J. G. Swoboda, Prof. S. Walker Department of Microbiology and Molecular Genetics Harvard Medical School Boston, MA 02115 (USA) Fax: (+1)617-738-7664 E-mail : suzanne_walker@hms.harvard.edu [b] Dr. C. J. Noren New England Biolabs Ipswich, MA 01938 (USA) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.