Yiwei Huang

C‐Terminal Incorporation of Bio‐Orthogonal Azide Groups into a Protein and Preparation of Protein–Oligodeoxynucleotide Conjugates by CuI‐Catalyzed Cycloaddition

Protein–oligodeoxynucleotide conjugates can be utilized for a variety of applications, like the preparation of synthetic enzymes, gene therapy, electrical DNA microarrays, molecular-scale devices, and the development of immunological assays. A number of protein conjugation techniques have been developed; however, these are usually based on the reaction with functional groups common in biological systems. They therefore lead to random chemical modification and functional heterogeneity of the products. 8] Known bio-orthogonal conjugation reactions, such as Staudinger ligations, ketone/aldehyde-hydrazine reactions, and Cu-catalyzed [3+2] cycloadditions, allow regioand chemoselective modification of proteins, and are especially attractive for construction of protein microarrays, for which function can be significantly altered by protein orientation on the solid surface. 14] Recently we reported the application of an E. coli in vitro transcription–translation coupled system (IVT system) to the site-specific modification of proteins. This was achieved by deactivation of release factor 1 (RF1) by using polyclonal antibodies that led to puromycin incorporation into the C terminus of esterase 2 (EST2). The attachment of nucleotides to the 5’-OH of puromycin was also possible. However, when more then six nucleotides were added to the 5’-OH of puromycin, the reaction yield strongly decreased. This hindered the preparation of protein conjugates with oligodeoxynucleotide (ODN) sufficiently long for specific hybridization. Here, we describe an alternative and equally efficient method for C-terminal modification of proteins by using an E. coli IVT system for incorporation of a bio-orthogonal azide group. With this system 5’-alkyne modified ODNs can be conjugated specifically to the C terminus of a polypeptide chain by Cu-catalyzed cycloaddition via a puromycin linker. Electrochemical detection of protein–ODN conjugates was realized by hybridization to complementary ODNs immobilized on a gold electrode microarray. 2’-Deoxy-cytidylyl-(3’!5’)-puromycin (1; Figure 1A) modified by an NH2 linker at the N4 position of cytidine was used as a starting material for further modifications. For this, N-hydroxy-

Peptide-Bond Synthesis on the Ribosome : No Free Vicinal Hydroxy Group Required on the Terminal Ribose Residue of Peptidyl-tRNA

The synthesis of a peptide bond is a reaction of central importance for biology. Despite remarkable progress in the last decade in the elucidation of ribosome structure, the catalytic mechanism of this reaction, which takes place in the peptidyltransferase center of the ribosome, remains unclear. According to the currently accepted view, peptide transfer is an entropically driven reaction in the protein-free active site formed by 23S RNA and bound 3’ tails of peptidyland aminoacyl-tRNA molecules. Peptide-bond formation occurs through nucleophilic attack of the a-amino group of aminoacyl-tRNA at the carbonyl group of peptidyl-tRNA with the formation of a tetragonal transition state. In this process, a proton is transferred from the a-amino group to the 3’-OH group of the peptidyl-tRNA. Free vicinal OH groups at the 3’ end of the aminoacyland peptidyl-tRNA are potential proton donors. Therefore, positional isomers of aminoacyland peptidyl-tRNA molecules have been studied extensively with respect to the attachment of the acyl residue to the 2’or 3’-OH group and requirements for the free vicinal OH groups during translation. For peptide transfer by a peptidyltranferase to occur, both the aminoacyl residue in the A site and the peptidyl residue in the P site have to be attached to the 3’-OH group of the terminal adenosine residue. The absence of the vicinal 2’-OH group in the aminoacyl-tRNA does not impair peptide transfer; however, the role of the 2’-OH group in the peptidyl-tRNA remains unclear. Peptidyltransferase activity measured by in vitro assays with tRNA fragments or puromycin as models for aminoacyland peptidyl-tRNA, or in assays in which short oligonucleotides were used as mRNA substitutes (again not a complete tRNA system), was inhibited by the absence of the 2’-OH peptidyl analogues bound in the P site. 10] However, in assays in which complete peptidyl-tRNA-2’dA and long mRNA were used, the peptidyltransferase tolerated the absence of the 2’-OH group. To resolve this discrepancy, we tested the activity of suppressor tRNA-2’dA in vitro by translating a complete mRNA of esterase 2 from Alicyclobacillus acidocaldarius with a nonsense UAG codon 155 and RF2-dependent termination codons (RF2= release factor 2). Codon 155 codes for a serine residue, an essential member of the catalytic triad, in esterase 2. Only the suppression of UAG155 by Ser-tRNA enables the synthesis of the active esterase. In the absence of Ser-tRNA, premature termination, frame shifting, or suppression with endogenous aminoacyl-tRNA occurs. Premature termination can be suppressed almost completely by the removal of release factor 1 (RF1) from the in vitro translation mixture. This assay, previously described for tRNA-A (aminoacyltRNA), provides a means to test the activity of tRNA-2’dA in the elongation cycle. If the mechanism for “substrate-assisted catalysis” involving the 2’-OH group during peptide transfer (Scheme 1 A) is correct, the replacement of tRNA-A with tRNA-2’dA should prevent the in vitro synthesis of esterase 2.

Efficient suppression of the amber codon in E. coli in vitro translation system

An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNASer(CUA) was monitored by determination of the full‐length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild‐type protein.

The esterase from Alicyclobacillus acidocaldarius as a reporter enzyme and affinity tag for protein biosynthesis.

Esterase from thermophilic bacteria Alicyclobacillus acidocaldarius can be produced up to 200 μg/ml by coupled in vitro transcription/translation system derived from Escherichia coli. The synthesized thermostable enzyme can be determined by photometrical and fluorescent assays at least up to 10−8 M concentration or by activity staining in the polyacrylamide gels. Enhanced green fluorescence protein‐esterase fusion protein was bound to a matrix with immobilized esterase inhibitor and purified by affinity chromatography. Thus, the esterase is suited as a reporter enzyme to monitor the expression of polypeptides coupled to its N‐terminus and simultaneously, as a cleavable tag for polypeptide purification.

Ligand‐Directed Immobilization of Proteins through an Esterase 2 Fusion Tag Studied by Atomic Force Microscopy

Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand‐directed immobilization of esterase‐linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin‐t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand–esterase interaction allowed specific attachment of exportin‐t and resulted in high‐resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin‐t in forming a quaternary complex with tRNA and the GTPase Ran‐GTP, and the dimension changes before and after complex formation were also determined by AFM.