Synthesis of Polyester by Means of Genetic Code Reprogramming

Ohta, Atsushi; Murakami, Hiroshi; Higashimura, Eri; Suga, Hiroaki

doi:10.1016/j.chembiol.2007.10.015

Cited by 138 publications

(120 citation statements)

References 29 publications

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“…Preparation of Flexizyme (dFx, eFx) and mycobacteriophage L5 (ML)-derived ML-tRNA Asn cta (tRNA sup ) were done using the same protocol. First, double-stranded DNA templates encoding the RNA species and a N-terminal T7 promotor sequence (18,19) 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%

Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

Öjemalm

Higuchi

Lara

et al. 2016

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

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Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔG app ) of "snorkeling" of charged amino acids toward the lipid-water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔG app . These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells. Using a cotranslational insertion assay, we previously measured the contribution of each of the 20 natural amino acids to the membrane integration efficiency of model transmembrane segments, and derived a "biological" hydrophobicity scale that assigns an apparent free-energy of membrane integration, ΔG aaðiÞ, j app , to each amino acid of type i [aa(i) = A, C, D, E, . . ., W, Y] as a function of its position j within a transmembrane α-helix (2). To further probe the physicochemical basis for membrane insertion, we also analyzed a series of aliphatic and aromatic nonproteinogenic amino acids, using the same insertion assay (3). This approach made it possible to quantitate the "hydrophobic effect" during membrane protein insertion, giving nonpolar solvation energy parameter values of -10 cal/(mol·Å 2 ) and -7 cal/(mol·Å 2 ) for aliphatic and aromatic surface area, respectively.Here, we extend our analysis of nonproteinogenic amino acids to analogs of the positively charged amino acids Arg and Lys. We provide estimates of the influence on the apparent free energy of membrane integration (ΔG app ) of "snorkeling" of charged amino acids toward the lipid-water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone H-bonds on ΔG app .

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Section: Methodsmentioning

confidence: 99%

Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

Öjemalm

Higuchi

Lara

et al. 2016

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

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“…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%

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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“…All oligonucleotides were purchased from Operon Biotechnologies. Flexizyme and tRNA molecules were synthesized using the same procedure as previously described (Murakami et al 2006;Ohta et al 2007;Goto et al 2008). …”

Section: Methodsmentioning

confidence: 99%

“…Accordingly, we decided to use the well-known fact that D aa are generally poor substrates in the elongation event using the amber suppression method (Noren et al 1989;Bain et al 1991;Ellman et al 1992;Starck et al 2003;Tan et al 2004;Murakami et al 2006). Since the flexizyme system is able to afford the D aa-tRNA molecules for initiation and elongation (charged onto tRNA fMet CAU and tRNA AsnE-2 CUA [Ohta et al 2007], respectively) with exactly the same quality, we should be able to verify the occurrence of racemization by running the peptide synthesis with the (Fig. 5A).…”

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confidence: 99%

Initiating translation with D-amino acids

Murakami¹,

Suga²

2008

RNA

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Here we report experimental evidence that the translation initiation apparatus accepts D-amino acids ( D aa), as opposed to only L-methionine, as initiators. Nineteen D aa, as the stereoisomers to their natural L-amino acids, were charged onto initiator tRNA fMet CAU using flexizyme technology and tested for initiation in a reconstituted Escherichia coli translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to D aa. Remarkably, all D aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with D aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding

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Synthesis of Polyester by Means of Genetic Code Reprogramming

Cited by 138 publications

References 29 publications

Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

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

Initiating translation with D-amino acids

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