Templated native chemical ligation: peptide chemistry beyond protein synthesis

Vázquez, Olalla; Seitz, Oliver

doi:10.1002/psc.2602

Cited by 43 publications

(31 citation statements)

References 121 publications

(138 reference statements)

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“…Because the composition of phospholipids controls the physical properties of the resulting membranes, synthetic phospholipid remodeling could be used to gain control over the function and form of artificial membranes, for instance by enabling dynamic changes in vesicle morphology. Here, we demonstrate efficient de novo generation and remodeling of phospholipid vesicles at neutral pH through application of the native chemical ligation (NCL) (13)(14)(15)(16)(17)(18) and reversible native chemical ligation (RNCL) (19) (Fig. 1 B and C and SI Appendix, Figs.…”

mentioning

confidence: 91%

Nonenzymatic biomimetic remodeling of phospholipids in synthetic liposomes

Brea

Rudd

Devaraj

2016

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

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Cell membranes have a vast repertoire of phospholipid species whose structures can be dynamically modified by enzymatic remodeling of acyl chains and polar head groups. Lipid remodeling plays important roles in membrane biology and dysregulation can lead to disease. Although there have been tremendous advances in creating artificial membranes to model the properties of native membranes, a major obstacle has been developing straightforward methods to mimic lipid membrane remodeling. Stable liposomes are typically kinetically trapped and are not prone to exchanging diacylphospholipids. Here, we show that reversible chemoselective reactions can be harnessed to achieve nonenzymatic spontaneous remodeling of phospholipids in synthetic membranes. Our approach relies on transthioesterification/ acyl shift reactions that occur spontaneously and reversibly between tertiary amides and thioesters. We demonstrate exchange and remodeling of both lipid acyl chains and head groups. Using our synthetic model system we demonstrate the ability of spontaneous phospholipid remodeling to trigger changes in vesicle spatial organization, composition, and morphology as well as recruit proteins that can affect vesicle curvature. Membranes capable of chemically exchanging lipid fragments could be used to help further understand the specific roles of lipid structure remodeling in biological membranes.L iving organisms carry out the de novo synthesis of phospholipid membranes, in part, by using membrane-bound acyltransferases and reactive thioester precursors ( Fig. 1A) (1). Subsequently, de novo synthesized phospholipid membranes can be remodeled by acyltransferases and transacylases in the presence of different lysolipid, phospholipid, and fatty acid precursors ( Fig. 1A) (2). Such natural mechanisms allow cells to fine-tune properties of biological membranes by changing the composition and organization of their constituent lipids and proteins (3). Membrane remodeling is an essential component of numerous cellular functions including the formation of organelles (4), division (5), trafficking (6), and signaling (7). Many experimental and theoretical studies have examined membrane remodeling due to enzymatically driven changes in lipid composition (8). For instance, the disruption of the transacylase activity of the enzyme tafazzin leads to alterations in the mitochondrial lipid cardiolipin and the disease known as Barth's syndrome (9).Model membranes composed of well-defined synthetic lipids are useful tools for studying questions related to membrane biology (10, 11). The importance of lipid remodeling in biology underscores the need for straightforward methods to remodel lipid composition in artificial model membranes. Unfortunately, nonenzymatic methods to exchange lysolipid fragments in synthetic vesicles have previously not been feasible. This is likely due to the high kinetic stability of phospholipid membranes, which do not readily exchange diacylphospholipid species between membranes (12). Because the composition of phospholipids c...

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confidence: 91%

Nonenzymatic biomimetic remodeling of phospholipids in synthetic liposomes

Brea

Rudd

Devaraj

2016

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

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“…Despite the r(CUG) n repeats ( n >5) adopting their own secondary hairpin structures, the triplet nucleic‐acid probe could still recognize the RNA targets and underwent template‐directed concatenation. Nucleic‐acid‐catalyzed oligomerization was previously demonstrated, in particular, by Lynn, Liu, and Seitz; however, none of these systems contained mutually reactive C‐terminal thioester and N‐terminal cysteine moieties in the same nucleic‐acid recognition module and none were designed to bind to biomedically relevant and structured RNA targets, such as CUG repeats. Overall, this work provides a proof of concept for the design of short nucleic‐acid probes for targeting RNA‐repeated expansions through template‐directed oligomerization, lending possibility for the development of therapeutic interventions for DM1 and other related neuromuscular and neurodegenerative disorders.…”

Section: Methodsmentioning

confidence: 99%

RNA‐Templated Concatenation of Triplet Nucleic‐Acid Probe

et al. 2018

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Template-directed synthesis offers several distinct benefits over conventional laboratory creation, including unsurpassed reaction rate and selectivity. Although it is central to many biological processes, such an approach has rarely been applied to the in situ synthesis and recognition of biomedically relevant target. Towards this goal, we report the development of a three-codon nucleic-acid probe containing a C-terminal thioester group and an N-terminal cysteine that is capable of undergoing template-directed oligomerization in the presence of an RNA target and self-deactivation in its absence. The work has implications for the development of millamolecular nucleic-acid probes for targeting RNA-repeated expansions associated with myotonic dystrophy type 1 and other related neuromuscular and neurodegenerative disorders.

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“…To overcome the issues we had encountered using genetic fusion approaches, we instead explored a technique to link two isolated proteins into fusion proteins via the use of a series of synthetic peptide linkers and protein ligation techniques ( Figure 2)specifically, NCL [48][49][50] and SML [43,[51][52][53][54][55]. We decided on these techniques as they result in the generation of a standard peptide bond at the site of conjunction, as opposed to the linkages that result from alternative techniques used for the generation of protein complexes [25][26][27]31].…”

Section: Snape In Vitro Protein Ligation Techniquementioning

confidence: 99%

SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides forin vitroapplications

Ulrich

Cryle

2016

J. Pept. Sci.

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Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

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Templated native chemical ligation: peptide chemistry beyond protein synthesis

Cited by 43 publications

References 121 publications

Nonenzymatic biomimetic remodeling of phospholipids in synthetic liposomes

Nonenzymatic biomimetic remodeling of phospholipids in synthetic liposomes

RNA‐Templated Concatenation of Triplet Nucleic‐Acid Probe

SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides forin vitroapplications

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