Site-Directed Mutagenesis with an Expanded Genetic Code

Mendel, David; Cornish, Virginia W.; Schultz, Peter G.

doi:10.1146/annurev.bb.24.060195.002251

Cited by 172 publications

(121 citation statements)

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“…Therefore, for site-speci®c variations additional codons would be required which are not available (and not required) in the protein biosynthesis machinery. However, recently such a method, which is based on an expanded genetic code, has been developed [56].…”

Section: Glycopeptide and Glycoprotein Synthesisðuse Of An Expanded Gmentioning

confidence: 99%

New aspects of glycoside bond formation

Schmidt¹,

Castro‐Palomino²,

Retz³

1999

Pure and Applied Chemistry

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Glycoside bond formation generally requires activation of the sugar at the anomeric center. To this end, anomeric oxygen exchange reactions, resulting in the Koenigs±Knorr method and variations or, alternatively, activation through retention of the anomeric oxygen, resulting in the trichloroacetimidate method and in the phosphite method, have been proposed. The successful application of the trichloroacetimidate method to the total synthesis of GPI anchors is particularly worth mentioning. a[2-3]-Sialylation can be based on sialyl phosphites as glycosyl donors and on the nitrile effect for anomeric stereocontrol. This is exhibited for a preparative synthesis of ganglioside GM 2 which is required for tumor vaccine studies. For the generation of a[2-8]-linkage between neuraminic acid residues anchimeric assistance by a 3-thiocarbonyloxy group is introduced. The required sialyl donor can be ef®ciently prepared and a-linkage to 8-O-unprotected neuraminic acid derivatives is almost quantitative. The limitations of chemical glycopeptide synthesis encourage to employ the protein biosynthesis machinery in combination with an expanded genetic code. This is exhibited for the synthesis of glycosylated hARF-1 protein. GLYCOSIDE BOND FORMATIONÐGENERAL ASPECTSGlycoside bond formation in order to gain chemically de®ned oligosaccharides and glycoconjugates remains an important task, though generation of the anomeric linkage has seen years of dynamic progress, mainly based on highly reactive glycosyl donors, on versatile building block strategies, and on advanced protective-group design [1±3]. The endeavour was stimulated by the eminent role of carbohydrates and especially of glycoconjugates in various ®elds of modern biology [4,5]. A particularly prominent area in this regard is glycoconjugate synthesis.

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Section: Glycopeptide and Glycoprotein Synthesisðuse Of An Expanded Gmentioning

confidence: 99%

New aspects of glycoside bond formation

Schmidt¹,

Castro‐Palomino²,

Retz³

1999

Pure and Applied Chemistry

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“…Efforts have been made to use cell-free synthesis to expand the repertoire of ribosomal synthesis to include noncoded amino acids as building blocks (6,7). These attempts to incorporate other amino acids have had very limited successobtaining adequate amounts of pure protein from the cell-free translation systems can be a significant challenge (8), and many unnatural amino acids are simply not compatible with ribosomal polypeptide synthesis (9).…”

Section: Introduction: Protein Science In the Postgenome Eramentioning

confidence: 99%

Synthesis of Native Proteins by Chemical Ligation

Dawson

Kent²

2000

Annu. Rev. Biochem.

1,583

1,526

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Key Words chemical protein synthesis, thioester, protein, peptide, solid phase synthesis, polymer-supported synthesis, protein engineering s Abstract In just a few short years, the chemical ligation of unprotected peptide segments in aqueous solution has established itself as the most practical method for the total synthesis of native proteins. A wide range of proteins has been prepared. These synthetic molecules have led to the elucidation of gene function, to the discovery of novel biology, and to the determination of new three-dimensional protein structures by both NMR and X-ray crystallography. The facile access to novel analogs provided by chemical protein synthesis has led to original insights into the molecular basis of protein function in a number of systems. Chemical protein synthesis has also enabled the systematic development of proteins with enhanced potency and specificity as candidate therapeutic agents.

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“…These studies have typically relied on total chemical synthesis methods (13) or specialized in vitro translation techniques (14) to incorporate amide-to-ester bond mutations into the polypeptide backbone of proteins and modulate the hydrogen-bonding properties of a protein's polypeptide backbone. The structural and thermodynamic consequences of introducing such amide-to-ester-bond mutations into ␤-sheet and ␣-helical regions of different protein folds have been reported (3)(4)(5)(6)(7)(8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

Conserved thermodynamic contributions of backbone hydrogen bonds in a protein fold

Wang

Wales

Fitzgerald

2006

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

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Backbone-backbone hydrogen-bonding interactions are a ubiquitous and highly conserved structural feature of proteins that adopt the same fold (i.e., have the same overall backbone topology). This work addresses the question of whether or not this structural conservation is also reflected as a thermodynamic conservation. Reported here is a comparative thermodynamic analysis of backbone hydrogen bonds in two proteins that adopt the same fold but are unrelated at the primary amino acid sequence level. With amide-to-ester bond mutations introduced by total chemical synthesis methods, the thermodynamic consequences of backbonebackbone hydrogen-bond deletions at five different structurally equivalent positions throughout the ␤-␣-␣ fold of Arc repressor and CopG were assessed. The ester bond-containing analogues all folded into native-like three-dimensional structures that were destabilized from 2.5 to 6.0 kcal͞(mol dimer) compared with wild-type controls. Remarkably, the five paired analogues with amide-to-ester bond mutations at structurally equivalent positions were destabilized to exactly the same degree, regardless of the degree to which the mutation site was buried in the structure. The results are interpreted as evidence that the thermodynamics of backbone-backbone hydrogen-bonding interactions in a protein fold are conserved.protein mutagenesis ͉ chemical synthesis T he 30,000-plus high-resolution protein structures that have been solved to date can be grouped, according to their overall backbone topology, into a remarkably small number of protein folds (Ϸ800) (1, 2). The apparent existence of such a small number of naturally occurring protein folds has meant that a surprisingly large number of proteins, which are unrelated at the amino acid sequence level, adopt the same protein fold (i.e., fold into three-dimensional structures with the same backbone topology). Thus, nature appears to have used a number of different combinations of chemical interactions to stabilize its protein folds. However, one set of chemical interactions that are structurally conserved within a protein fold is the set of backbone-backbone hydrogen-bonding interactions that help define it. This structural conservation raises a fundamental question of whether or not the thermodynamics of backbone-backbone hydrogen-bonding interactions are also conserved in a protein fold. Such fundamental knowledge about the detailed molecular interactions that guide protein-folding reactions stands to impact a wide variety of research areas from drug discovery to theories of human evolution.The relative contributions of different backbone-backbone hydrogen-bonding interactions in protein-folding reactions have been explored in a series of recent studies on several different protein systems (3-12). These studies have typically relied on total chemical synthesis methods (13) or specialized in vitro translation techniques (14) to incorporate amide-to-ester bond mutations into the polypeptide backbone of proteins and modulate the hydrogen-bonding properties o...

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Site-Directed Mutagenesis with an Expanded Genetic Code

Cited by 172 publications

References 0 publications

New aspects of glycoside bond formation

New aspects of glycoside bond formation

Synthesis of Native Proteins by Chemical Ligation

Conserved thermodynamic contributions of backbone hydrogen bonds in a protein fold

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