Spontaneous Re-formation of a Broken Peptide Chain

Dyckes, Douglas F.; Creighton, Thomas E.; Sheppard, R. C.

doi:10.1038/247202a0

Cited by 54 publications

(20 citation statements)

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“…This effect was first reported by Sheppard and co-workers, 22 who noticed that pancreatic trypsin inhibitor fragments obtained by CNBr cleavage were able to spontaneously religate forming the original native peptide bond between them. The same effect was also observed for cytochrome C. 23 In both cases, the two protein fragments produced after the CNBr cleavage, were able to cooperatively refold thus bringing the homoserine lactone at the C-terminus of one of the fragments in close proximity of the α-amino group of the other fragment.…”

Section: Figure 1 Attaching Proteins To Solid Surfaces By Using a Prmentioning

confidence: 73%

Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

Camarero¹

2006

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Protein arrays are ideal tools for the rapid analysis of whole proteomes as well as for the development of reliable and cheap biosensors. The objective of this proposal is to develop a new ligand assisted ligation method based in the naturally occurring protein trans-splicing process. This method has been used for the generation of spatially addressable arrays of multiple protein components by standard micro-lithographic techniques. Key to our approach is the use of the protein trans-splicing process. This naturally occurring process allows the development of a truly generic and highly efficient method for the covalent attachment of proteins through its C-terminus to any solid support. This technology has been used for the creation of protein chips containing several virulence factors from the human pathogen Y. pestis. IntroductionMany experimental techniques in biology and biophysics, and applications in diagnosis and drug discovery, require proteins immobilized on solid substrates. [1][2][3] In fact, the concept of arrays of proteins attached to a solid support has attracted increasing attention over the last three years due to the sequencing of several genomes, including the human genome. When a genome has been deciphered, the daunting task of determining the function of each protein encoded in the genome still remains. Protein arrays can be used easily for such analysis in a parallel fashion.2,4 Another powerful application employs ordered nanometric arrays of proteins as nucleation templates for protein crystallization or for structural studies. Recent advances in nanoprinting techniques have allowed the creation of sub-micrometer arrays of proteins. 5,6 All these applications demonstrate the use of protein arrays and also highlight the need for methods able to attach proteins in a well defined and ordered way onto a solid supports.Various methods are available for attaching proteins to solid surfaces. Most rely on non-specific adsorption, 6,7 or on the reaction of chemical groups within proteins (mainly, amino and carboxylic acid groups) with surfaces containing complementary reactive groups. 8,9 In both cases the protein is attached to the surface in random orientations. The use of recombinant affinity tags addresses the orientation issue. However, in most cases the interactions of the tags are reversible and therefore not stable over the course of subsequent assays or require large mediator proteins.10,11 Methods for the chemoselective attachment of proteins to surfaces has been also developed recently by our group.12-15 These methods rely in the introduction of two unique and mutually reactive groups on the protein and the support surface. The reaction between these two groups usually gives rise to the selective attachment of the protein to the surface with total control over the orientation.14,15 However, these methods, although highly selective, rely on uncatalyzed pseudo-bimolecular reactions with little or not activation at all. This lack of activation means that the efficiency of these bim...

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Section: Figure 1 Attaching Proteins To Solid Surfaces By Using a Prmentioning

confidence: 73%

Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

Camarero¹

2006

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“…It would also imply the use of weak carboxyl activations to prevent random acylations. This ideal situation has recently been achieved in the semisynthesis of basic pancreatic trypsin inhibitor (1). A similar synthesis has subsequently been described for a horse heart cytochrome c analog (2).…”

Section: )mentioning

confidence: 97%

“…NE-protected cytochrome c (1). Cytochrome c (553 mg, 44.7 ,umol) was dissolved in water (20 ml) and dimethylformamide (DMF) (15 ml).…”

Section: )mentioning

confidence: 99%

“…RESULTS AND DISCUSSION The spontaneous reformation of a broken peptide chain in a rigid molecule (basic pancreatic trypsin inhibitor), as described by Dyckes et al (1), exemplified a potential new tool in peptide and protein chemistry. This was followed by the genuine intermolecular condensation of two separated fragments of horse heart cytochrome c to give the complete enzyme (2).…”

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confidence: 99%

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Semisynthetic horse heart [65-homoserine]cytochrome c from three fragments

Boon¹,

Tesser²,

Nivard³

1979

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

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“…Contemporaneous with this development was the observation of a protein splicing system operative without benefit of enzyme catalysis or an equilibrium-shifting environment (Dyckes et al, 1974). CNBr cleavage converts methionine to homoserine, which can exist as an internal ester when C-terminal in a peptide.…”

Section: Peptide Splicing In Protein Semisynthesismentioning

confidence: 99%

Untitled

1993

Protein Science

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The gradual accumulation of examples of protein splicing, in which a nested intervening sequence is spliced out of the interior of a polyprotein precursor, suggests that this curious phenomenon might prove to have universal phylogenetic distribution and biological significance. The known examples are reviewed, with the aim of establishing underlying patterns, and a generalized mechanism of autocatalytic protein splicing is proposed. The testable consequences of such a proposal and the possible evolutionary origins of the phenomenon are discussed.Keywords: autocatalysis; introns; molecular evolution; polyproteins; protein semisynthesis; protein splicing A significant recent event for protein science is the discovery of protein splicing, the excision of intervening peptide sequences in the generation of a mature protein product. Such a facility to break and religate peptide bonds has always been a prime goal of those seeking to engineer protein structure by semisynthetic methods, and in the past few years methods based in quasi-biological principles have evolved as the most commonly used approaches. Now we find that nature has been splicing proteins all along. The process, analogous to the RNA splicing of introns, is of particular fascination to those of us engaged in protein semisynthesis. Perhaps if we can decipher the underlying mechanisms those principles will be of use in semisynthetic strategies. But more importantly we are faced with a novel phenomenon, the understanding of which might be of general biological relevance and certainly will illuminate studies of protein structurefunction relationships and molecular evolution. We want to know how protein splicing works.My goal then is to review what is known about protein splicing from the relatively few examples yet at our disposal and attempt to relate that knowledge to the work done on protein fragment religation for semisynthetic

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Spontaneous Re-formation of a Broken Peptide Chain

Cited by 54 publications

References 12 publications

Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

Semisynthetic horse heart [65-homoserine]cytochrome c from three fragments

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