A highly flexible tRNA acylation method for non-natural polypeptide synthesis

Murakami, Hiroshi; Ohta, Atsushi; Ashigai, Hiroshi; Suga, Hiroaki

doi:10.1038/nmeth877

Cited by 387 publications

(442 citation statements)

References 15 publications

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“…1. A suppressor tRNA (tRNA sup ) is charged with the desired nonproteinogenic amino acid using the Flexizyme system (8,9) and is then added to an in vitro translation system programmed with an mRNA encoding an engineered version of the well-characterized membrane-protein leader peptidase (Lep). The Lep construct has two N-terminal transmembrane helices (TM1, TM2) and contains a hydrophobic test segment (H segment) flanked by two canonical Asn-X-Thr acceptors sites for N-linked glycosylation (G1, G2).…”

Section: Resultsmentioning

confidence: 99%

“…Preparations were done using the same protocol. First, double-stranded DNA templates encoding the RNA species and an N-terminal T7 promotor sequence (8,32) were generated by PCR extension of annealed overlapping oligonuclotides. DNA templates were then amplified by PCR using primers complementary to both ends of the templates, followed by phenol/chloroform extraction and ethanol precipitation.…”

Section: Methodsmentioning

confidence: 99%

“…For silica gel preparative TLC preparation, Merck precoated plates (silica gel 60 F 254 , Art 5744, 0.5 mm) were used. Cyanomethylester (CME) and 3,5-dinitrobenzylester derivatives were prepared using a previously described procedure (8,33). For general procedures of synthesis and characterization of compounds, see SI Text.…”

Section: Methodsmentioning

confidence: 99%

“…RNA was ethanol precipitated twice, once with a 0.1 M ethanolic AcONa solution (pH 5.2) and once with 95% ethanol. More detailed information can be found elsewhere (8).…”

Section: Methodsmentioning

confidence: 99%

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Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

Öjemalm

Higuchi

Jiang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid's contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal∕ðmol · Å 2 Þ, the solvation parameter for transfer from translocon to bilayer is 6-10 cal∕ðmol · Å 2 Þ, pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon.flexizyme | hydrophobicity | nonproteinogenic amino acid | transmembrane helix I n eukaryotic cells, membrane proteins destined for the plasma membrane and the various compartments along the endo-and exocytic pathways are synthesized by endoplasmic reticulum (ER)-bound ribosomes and cotranslationally integrated into the ER membrane in a process mediated by the Sec61 translocon complex; the homologous SecYEG translocon mediates membrane-protein integration into the inner membrane of prokaryotes (1, 2). Subsequent to membrane integration, membrane proteins fold and oligomerize in the ER and are then moved further along the secretory pathway by vesicular transport.During the membrane-integration step, hydrophobic segments in the translocating nascent polypeptide chain exit the Sec61 translocon through a lateral gate and become embedded in the surrounding lipid bilayer (3-5). Cotranslational, transloconmediated integration of transmembrane α-helices into the ER membrane sets the stage for all subsequent folding and oligomerization events and hence represents a critical step in the maturation of membrane proteins.In previous studies, we have provided quantitative data on the propensities of the 20 natural amino acids to promote the integration of transmembrane helices into the ER membrane and have shown that they depend both on hydrophobicity and on position within the helix (3, 6). Although the partitioning of transmembrane helices between the Sec61 translocon and the lipid membrane bears strong similarities to partitioning of solutes between water and lipid membranes, translocon-to-bilayer partitioning may not be equivalent to water-to-bilayer partitioning (7). Insights into the differences between the two partitioning processes might be revealed i...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…RNA was ethanol precipitated twice, once with a 0.1 M ethanolic AcONa solution (pH 5.2) and once with 95% ethanol. More detailed information can be found elsewhere (8).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

Öjemalm

Higuchi

Jiang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Consequently, this approach paves the way for investigation of the structural basis of the pathological threshold found in at least ten different poly‐Q diseases. The development of other orthogonal tRNA/synthetase pairs or the use of established synthetic tools, such as flexizyme25 or chemical aminoacylation,17 to load nonsense tRNA with natural amino acids could extend the described strategy for the exploration of other LCRs. In this way, this important but structurally elusive family of proteins can be investigated at high resolution to decipher the structural basis of crucial biological processes and to guide drug design.…”

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confidence: 99%

A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats

et al. 2018

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Homorepeat (HR) proteins are involved in key biological processes and multiple pathologies, however their high‐resolution characterization has been impaired due to their homotypic nature. To overcome this problem, we have developed a strategy to isotopically label individual glutamines within HRs by combining nonsense suppression and cell‐free expression. Our method has enabled the NMR investigation of huntingtin exon1 with a 16‐residue polyglutamine (poly‐Q) tract, and the results indicate the presence of an N‐terminal α‐helix at near neutral pH that vanishes towards the end of the HR. The generality of the strategy was demonstrated by introducing a labeled glutamine into a pathological version of huntingtin with 46 glutamines. This methodology paves the way to decipher the structural and dynamic perturbations induced by HR extensions in poly‐Q‐related diseases. Our approach can be extended to other amino acids to investigate biological processes involving proteins containing low‐complexity regions (LCRs).

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Overview of Cell‐Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

Chong

2014

CP Molecular Biology

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During early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owning to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from “traditional” E. coli, rabbit reticulocyte lysate and wheat germ extracts to recent insect and human cell extracts to defined systems reconstituted from purified recombinant components. Though each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This article provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology.

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A highly flexible tRNA acylation method for non-natural polypeptide synthesis

Cited by 387 publications

References 15 publications

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats

Overview of Cell‐Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

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