Genetic Encoding of a Photocaged Histidine for Light-Control of Protein Activity.

The use of light to control protein function is a critical tool in chemical biology. Here we describe the addition of a photocaged histidine to the genetic code. This unnatural amino acid becomes histidine upon exposure to light and allows for the optical control of enzymes that utilize active-site histidine residues. We demonstrate light-induced activation of a blue fluorescent protein and a chloramphenicol transferase. Further, we genetically encoded photocaged histidine in mammalian cells. We then used this approach in live cells for optical control of firefly luciferase and, Renilla luciferase. This tool should have utility in manipulating and controlling a wide range of biological processes.

Genetic encoding of isobutyryl-, isovaleryl-, and ß-hydroxybutryl-lysine in E. coli.

Here we report the synthesis and genetic encoding of the lysine post translational modifications, ß-hydroxybutyryl-lysine, isobutyryl-lysine and isovaleryl-lysine. The ability to obtain a homogenous protein samples with site-specific incorporation of these acylated lysine residues can serve as a powerful tool to study the biological role of lysine post translational modifications.

Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK.

Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) Nϵ -L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.

Development of Genetically Encoded Biosensors for Reporting the Methyltransferase-Dependent Biosynthesis of Semisynthetic Macrolide Antibiotics.

Clarithromycin is an improved semisynthetic analogue of the naturally occurring macrolide, erythromycin. The subtle modification of a methyl group on the C-6 hydroxyl group endows the molecule with improved acid stability and results in a clinically useful antibiotic. Here, we show that the effector specificity of the biosensor protein, MphR, can be evolved to selectively recognize clarithromycin and therefore report on the production of this molecule in vivo. In addition, a crystal structure of the evolved variant reveals the molecular basis for selectivity and provides a guide for the evolution of a new metabolic function using this biosensor.

Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids.

We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) Nε-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2-6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of Nε-Boc-l-Lysine (BocK) and Nε-propargyl-l-Lysine (PrK) by 2-5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.

Ubiquitin Chains Bearing Genetically Encoded Photo-Cross-Linkers Enable Efficient Covalent Capture of (Poly)ubiquitin-Binding Domains.

Ubiquitin-mediated signaling pathways regulate essentially every aspect of cell biology in eukaryotes. Ubiquitin receptors typically contain ubiquitin-binding domains (UBDs) that have the ability to recognize monomeric ubiquitin (Ub) and polymeric Ub (polyUb) chains. However, how signaling specificity is achieved remains poorly understood, and many of the UBDs that selectively recognize polyUb chains of particular linkages still need to be identified and characterized. Here we report the incorporation of a genetically encoded photo-cross-linker, p-benzoyl-l-phenylalanine (Bpa), into recombinant Ub and enzymatically synthesized polyUb chains. This allows photo-cross-linking (covalent bond formation) of monoUb and K48- and K63-linked diUb chains to UBDs. This approach provides a framework for understanding Ub cellular signaling through the capture and identification of (poly)Ub-binding proteins.

Development of Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic Biology.

Macrolides are a large group of natural products that display broad and potent biological activities and are biosynthesized by type I polyketide synthases (PKSs) and associated enzymatic machinery. There is an urgent need to access macrolides and unnatural macrolide derivatives for drug discovery, drug manufacture, and probe development. Typically, efforts to engineer the biosynthesis of macrolides and macrolide analogues in various microbial hosts are hampered by the complexity of macrolide biosynthetic pathways and our limited ability to rationally reprogram type I PKSs and post-PKS machinery. High-throughput approaches based on synthetic biology and directed evolution could overcome this problem by testing the function of large libraries of variants. Yet, methods that can identify mutant enzymes, pathways, and strains that produce the desired macrolide target are not generally available. Here we show that the promiscuous macrolide sensing transcription factor MphR is a powerful platform for engineering variants with tailored properties. We identified variants that displayed improved sensitivity toward erythromycin, tailored the inducer specificity, and significantly improved sensitivity to macrolides that were very poor inducers of the wild-type MphR biosensor. Designer macrolide biosensors should find broad utility and enable applications related to high-throughput synthetic biology and directed evolution of macrolide biosynthesis.

Genetic incorporation of 4-fluorohistidine into peptides enables selective affinity purification.

Due to the lowered pKa of 4-fluorohistidine relative to histidine, peptides and proteins containing this amino acid are potentially endowed with novel properties. We report here the optimized synthesis of 4-fluorohistidine and show that it can efficiently replace histidine in in vitro translation reactions. Moreover, peptides containing 6×-fluorohistidine tags are able to be selectively captured and eluted from nickel resin in the presence of his-tagged protein mixtures.

Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins.

Polyubiquitination, a critical protein post-translational modification, signals for a diverse set of cellular events via the different isopeptide linkages formed between the C terminus of one ubiquitin (Ub) and the ɛ-amine of K6, K11, K27, K29, K33, K48, or K63 of a second Ub. We assembled di-ubiquitins (Ub2) comprising every lysine linkage and examined them biochemically and structurally. Of these, K27-Ub2 is unique as it is not cleaved by most deubiquitinases. As this remains the only structurally uncharacterized lysine linkage, we comprehensively examined the structures and dynamics of K27-Ub2 using nuclear magnetic resonance, small-angle neutron scattering, and in silico ensemble modeling. Our structural data provide insights into the functional properties of K27-Ub2, in particular that K27-Ub2 may be specifically recognized by K48-selective receptor UBA2 domain from proteasomal shuttle protein hHR23a. Binding studies and mutagenesis confirmed this prediction, further highlighting structural/recognition versatility of polyubiquitins and the potential power of determining function from elucidation of conformational ensembles.

Genetic encoding of the post-translational modification 2-hydroxyisobutyryl-lysine.

We report the synthesis and genetic encoding of a recently discovered post-translational modification, 2-hydroxyisobutyryl-lysine, to the genetic code of E. coli. The production of homogeneous proteins containing this amino acid will facilitate the study of modification in full-length proteins.

Rational protein sequence diversification by multi-codon scanning mutagenesis.

A new method for protein sequence diversification is based on generating random codon mutations to an encoding DNA. This allows for the scanning of user-defined amino acid changes to any protein of interest, and is an alternative to traditional directed evolution strategies. This chapter describes the procedures required to apply this technology to any protein of interest. The resulting libraries can then be screened for new or improved protein function.

Experimental methods for scanning unnatural amino acid mutagenesis.

Site-specific incorporation of unnatural amino acids into proteins in vivo relies on the genetic reassignment of nonsense or quadruplet codons. Here, we describe a general procedure for the random introduction of these codons into open reading frames resulting in protein libraries that are scanned with unnatural amino acid residues. These libraries can enable large-scale mutagenesis experiments aimed at understanding and improving protein function.

A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons.

Random mutagenesis followed by selection or screening is a commonly used strategy to improve protein function. Despite many available methods for random mutagenesis, nearly all generate mutations at the nucleotide level. An ideal mutagenesis method would allow for the generation of 'codon mutations' to change protein sequence with defined or mixed amino acids of choice. Herein we report a method that allows for mutations of one, two or three consecutive codons. Key to this method is the development of a Mu transposon variant with asymmetric terminal sequences. As a demonstration of the method, we performed multi-codon scanning on the gene encoding superfolder GFP (sfGFP). Characterization of 50 randomly chosen clones from each library showed that more than 40% of the mutants in these three libraries contained seamless, in-frame mutations with low site preference. By screening only 500 colonies from each library, we successfully identified several spectra-shift mutations, including a S205D variant that was found to bear a single excitation peak in the UV region.

Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme.

Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the ubiquitin field. Here, we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked diubiquitin, and a mixed-linkage Lys33,Lys11-linked triubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.

Photochemical control of bacterial signal processing using a light-activated erythromycin.

Bacterial cells control resistance to the macrolide antibiotic erythromycin using the MphR(A) repressor protein. Erythromycin binds to MphR(A), causing release of the PmphR promoter, activating expression of the 2'-phosphotransferase Mph(A). We engineered the MphR(A)/promoter system to, in conjunction with a light-activatable derivative of erythromycin, enable photochemical activation of gene expression in E. coli. We applied this photochemical gene switch to the construction of a light-triggered logic gate, a light-controlled band-pass filter, as well as spatial and temporal control of gene expression.

Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups.

E2 enzymes catalyze the ATP-dependent polymerization of polyubiquitin chains which function as molecular signals in the regulation of numerous cellular processes. Here we present a method that uses genetically encoded unnatural amino acids to halt and re-start ubiquitin polymerization providing access to natural-linkage, precision-length ubiquitin chains that can be used for biochemical, structural, and dynamics studies.

Condensed E. coli cultures for highly efficient production of proteins containing unnatural amino acids.

Current biosynthetic methods for producing proteins containing site-specifically incorporated unnatural amino acids are inefficient because the majority of the amino acid goes unused. Here we present a universal approach to improve the efficiency of such processes using condensed Escherichia coli cultures.