Expressed protein ligation: a resourceful tool to study protein structure and function

Berrade, Luis; Camarero, Julio A.

doi:10.1007/s00018-009-0122-3

Cited by 67 publications

(47 citation statements)

References 128 publications

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“…When we began this study, the lack of knowledge of a specific N-terminal acetyltransferase acting on ␣-syn, and the lack of chemical methods to specifically acetylate the N terminus of recombinant proteins, led us to rely on a protein semisynthesis approach based on expressed protein ligation (EPL) (1), which has been recently applied by our group to prepare N-terminal monoubiquitylated and C-terminal phosphorylated forms of ␣-syn (32,33). Later, we applied a recombinant expression strategy based on co-expression of ␣-syn and NatB, which generated quantitatively acetylated ␣-syn based on mass spectroscopic analysis.…”

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confidence: 99%

Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells

Fauvet

Fares

Samuel

et al. 2012

Journal of Biological Chemistry

164

157

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Background: How N-terminal acetylation affects the structure and function of ␣-syn remains unknown. Results: N-terminally acetylated and WT ␣-syn are unfolded monomers and exhibit similar aggregation and cellular properties. Conclusion: ␣-syn N-terminal acetylation does not dramatically affect its structure or oligomerization state in vitro and in intact cells. Significance: Recombinant nonacetylated or N ␣ -acetylated ␣-syn remains suitable for ␣-syn biophysical studies.

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mentioning

confidence: 99%

Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells

Fauvet

Fares

Samuel

et al. 2012

Journal of Biological Chemistry

164

157

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“…In this reaction, a fully unprotected peptide with an ␣-thioester at the C terminus reacts chemoselectively under neutral aqueous conditions with another unprotected peptide containing an N-terminal Cys residue to form a native peptide bond (19). It is also well established that when these two reactive groups are located in the same linear polypeptide precursor, the chemical ligation can proceed in an intramolecular fashion, therefore giving rise to a backbone-cyclized polypeptide (20). This reaction has been successfully used for the chemical synthesis of circular peptides and small protein domains (20).…”

Section: Backbone Cyclization Using Expressed Protein Ligationmentioning

confidence: 99%

“…It is also well established that when these two reactive groups are located in the same linear polypeptide precursor, the chemical ligation can proceed in an intramolecular fashion, therefore giving rise to a backbone-cyclized polypeptide (20). This reaction has been successfully used for the chemical synthesis of circular peptides and small protein domains (20).…”

Section: Backbone Cyclization Using Expressed Protein Ligationmentioning

confidence: 99%

“…This technology, called EPL, has provided access to a multitude of chemically engineered proteins, including backbone-cyclized polypeptides (see Ref. 20 for a recent review).…”

Section: Backbone Cyclization Using Expressed Protein Ligationmentioning

confidence: 99%

“…Gly and Ala residues react faster, whereas ␤-branched residues react slower and produce product in lower yields. Furthermore, the engineered inteins most commonly used for the generation of polypeptide ␣-thioesters have also been shown to be compatible with most amino acids upstream of the cleavage site (20).…”

Section: Backbone Cyclization Using Intein-mediated Protein Trans-splmentioning

confidence: 99%

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Biological Synthesis of Circular Polypeptides

Aboye

Camarero

2012

Journal of Biological Chemistry

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Here, we review the use of different biochemical approaches for biological synthesis of circular or backbone-cyclized proteins and peptides. These methods allow the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Protein circularization can significantly impact protein engineering and research in protein folding. Basic polymer theory predicts that circularization should lead to a net thermodynamic stabilization of a folded protein by reducing the entropy associated with the unfolded state. Protein cyclization also provides a valuable tool for exploring the effects of topology on protein folding kinetics. Furthermore, the biological production of cyclic polypeptides makes possible the production of cyclic polypeptide libraries. The generation of such libraries, which was previously restricted to the domain of synthetic chemists, now offers biologists access to highly diverse and stable molecular libraries for probing protein structure and function.Protein engineering is usually defined as the modification of the primary sequence of a protein to change in a defined way its three-dimensional structure and biological function. Site-specific mutagenesis using either recombinant DNA technology or chemical synthesis has been successfully applied to numerous proteins. Another useful approach to protein engineering involves changing backbone topology. Backbone cyclization or circularization (i.e. the covalent linkage of the N-terminal amino and C-terminal carboxylic groups) is a powerful tool for the study and manipulation of protein structure and function. The introduction of a covalent bond between the N and C termini of a protein provides a more stable topological constraint than the use of disulfide bonds. Hence, in proteins whose N and C termini are close in the native structure (a surprisingly common feature in folded proteins, particularly in single-domain proteins (1)), backbone cyclization should stabilize the protein fold by reducing the backbone entropy associated with the unfolded state of a protein. Backbone cyclization also has the added benefit of making proteins more resistant to proteolytic degradation, in particular against exoproteases (2, 3), which should increase both the in vitro stability of industrial enzymes and the in vivo stability of proteins with interesting pharmacological properties. In addition, protein cyclization provides a valuable tool for exploring fundamental questions in protein folding, in particular how topology affects the kinetics of protein folding (4).Backbone peptide cyclization has also been widely used in bioorganic and medicinal chemistry to improve the biochemical and biophysical properties of flexible and labile peptides in the development of peptide-based drug candidates (2, 3). The cyclization of flexible linear peptides reduces their conformational freedom and creates constrained structural frameworks that often confer high receptor binding affinity, specificity, and enhanced stability (2, 3).Backbon...

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Amide‐Forming Ligation Reactions

2019

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One of the long‐standing pursuits of synthetic organic chemists is the development of chemical methods for the synthesis of peptides and proteins as tools for understanding biological processes and providing therapeutic solutions to human diseases. The advent of chemoselective ligation reactions for the chemical synthesis of peptides and proteins has enabled synthetic chemists to realize this long‐held dream. In an amide‐forming ligation reaction, two uniquely placed functional groups within the peptide allow peptide or protein segments to be joined in a chemoselective manner with an amide bond. In this chapter, some of the early methods based on principles of the capture/rearrangement strategy for ligation of peptides are catalogued, followed by some of the most versatile and well‐established contemporary methods for the preparation of linear/cyclic peptides and proteins such as the native chemical ligation (NCL) and the α‐ketoacid–hydroxylamine (KAHA) ligation. The chapter concludes with some of the most promising new generation ligation methods such as the potassium acyltrifluoroborate–hydroxylamine (KAT) ligation. The tremendous progress in the past two decades in the development of several ligation methods that rely on a pair of unique functional groups is set to expand the horizons of fully functional proteins and multi‐protein assemblies that are purely synthetically prepared. Although contemporary ligation reactions do not match the speed and efficiency at which proteins are assembled in biological systems, the repertoire of chemoselective amide‐forming ligation methods has already enabled synthetic protein chemistry to surpass biology in terms of chemical and structural diversity.

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Expressed protein ligation: a resourceful tool to study protein structure and function

Cited by 67 publications

References 128 publications

Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells

Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells

Biological Synthesis of Circular Polypeptides

Amide‐Forming Ligation Reactions

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