Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography

Genetically encoded caged amino acids can be used to control the dynamics of protein activities and cellular localization in response to external cues. In the present study, we revealed the structural basis for the recognition of O-(2-nitrobenzyl)-L-tyrosine (oNBTyr) by its specific variant of Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (oNBTyrRS), and then demonstrated its potential availability for time-resolved X-ray crystallography. The substrate-bound crystal structure of oNBTyrRS at a 2.79 Å resolution indicated that the replacement of tyrosine and leucine at positions 32 and 65 by glycine (Tyr32Gly and Leu65Gly, respectively) and Asp158Ser created sufficient space for entry of the bulky substitute into the amino acid binding pocket, while Glu in place of Leu162 formed a hydrogen bond with the nitro moiety of oNBTyr. We also produced an oNBTyr-containing lysozyme through a cell-free protein synthesis system derived from the Escherichia coli B95. ΔA strain with the UAG codon reassigned to the nonnatural amino acid. Another crystallographic study of the caged protein showed that the site-specifically incorporated oNBTyr was degraded to tyrosine by light irradiation of the crystals. Thus, cell-free protein synthesis of caged proteins with oNBTyr could facilitate time-resolved structural analysis of proteins, including medically important membrane proteins

Cell-Free Protein Synthesis Using S30 Extracts from <i>Escherichia coli</i> RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of &gt;90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements

Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody–antigen interaction

<div><p>Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody–antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059–152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355^EGFR, Gln384^EGFR, H409^EGFR, and Lys465^EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.</p></div

Structure of a putative trans-editing enzyme for prolyl-tRNA synthetase from Aeropyrum pernix K1 at 1.7 Å resolution

The three-dimensional structure of the APE2540 protein from A. pernix K1 has been determined by the multiple anomalous dispersion method at 1.7 Å resolution. The structure includes two monomers in the asymmetric unit and shares structural similarity with the YbaK protein or cysteinyl-tRNAPro deacylase from H. influenzae

SDS-PAGE analysis of cell-free synthesized 059-152-Fv and 059-152-Fab.

<p>(A) 059-152-Fv was synthesized under a series of different concentrations (0, 0.2, and 0.4 mg/ml) of DsbC, as indicated. Total (T) and soluble (S) fractions of the internal solution were analyzed by reducing SDS-PAGE. (B) Purified 059-152-Fv was analyzed by reducing and non-reducing SDS-PAGE. The yields (mg per 1 ml internal solution) of partially purified Fv are indicated under each lane of the non-reducing SDS polyacrylamide gel image. (C) 059-152-Fab was synthesized in the presence of 0, 0.2, 0.4, and 0.8 mg/ml of DsbC, as indicated. (D) Purified 059-152-Fab was analyzed by reducing and non-reducing SDS-PAGE. The yields (mg per 1 ml internal solution) of partially purified Fab are indicated under each lane of the non-reducing SDS polyacrylamide gel image. BG: cell-free synthesis without template DNA. VH: cell-free synthesis of VH without DsbC. VL: cell-free synthesis of VL without DsbC. VHCH1: cell-free synthesis of VHCH1 without DsbC. L chain: cell-free synthesis of light chain without DsbC. Gels were stained with CBB.</p

SDS-PAGE analysis of site-specific fluorescent-labeled 059-152-Fv and 059-152-Fab.

<p>Reducing and non-reducing SDS-PAGE analysis of 059-152-Fv (A) and 059-152-Fab (B). (lane 1) 059-152-Fv, (lane 2) AzF-incorporated 059-152-Fv, (lane 3) Alexa-488 conjugated 059-152-Fv, (lane 4) 059-152-Fab, (lane 5) AzF-incorporated 059-152-Fab, and (lane 6) Alexa-488 conjugated 059-152-Fab. Fluorescent images and CBB-stained images were acquired from the same gels.</p

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers

光でイオンを輸送する膜タンパク質の巧妙な仕組み --XFELが捉えた光駆動型イオンポンプロドプシンの構造変化--. 京都大学プレスリリース. 2022-02-28.Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p