A Designed Peptide Ligase for Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues

Jackson, David Y.; Burnier, John; Quan, Clifford; Stanley, Mark; Tom, Jeffrey; Wells, James A.

doi:10.1126/science.7939659

Cited by 297 publications

(233 citation statements)

References 35 publications

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“…The artificial mutants exhibited an unchanged kcat but with greatly modified pH profiles. [65] This result is consistent with these histidines delivering H-bonds for nucleophile orientation as well as for GABC.…”

Section: Desolvation -Activate the Nucleophile And The Electrophilesupporting

confidence: 82%

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

et al. 2017

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Abstract:The 1994 structure of a transition state analog with AlF4 -and GDP complexed to G1α, a small G protein, heralded a new field of research into structure and mechanism of enzymes that manipulate transfer of the phosphoryl (PO3 -) group. The list of enzyme structures that embrace metal fluorides, MFx, as ligands that imitate either the phosphoryl group or a phosphate, is now growing at over 80 per triennium. They fall into three distinct geometrical classes: (i) Tetrahedral complexes, based on BeF3 -, mimic ground state phosphates; (ii) Octahedral complexes, primarily based on AlF4 -, mimic "in-line" anionic transition state for phosphoryl transfer; and (iii) Trigonal bipyramidal complexes, represented by MgF3 -and putative AlF3 0 moieties, additionally mimic the tbp geometry of the transition state. The interpretation of these structures provides a deeper mechanistic understanding of the behavior and manipulation of phosphate monoesters in molecular biology. This review provides a comprehensive overview of these structures, their uses, and their computational development. It questions the identification of AlF3 0 and MgF4 = as tbp species in protein complexes and discusses the relevance of physical organic chemistry and water-based model studies for understanding phosphoryl group transfer in enzymes. It describes two roles for amino acid side-chains that mediate proton transfers during phosphoryl transfer, based on the analysis of protein/MFx structures. First, they deploy hydrogen bonding to neutral oxygen nucleophiles so as to orientate them for correct orbital overlap with the electrophilic phosphorus center. Secondly, they behave as classical general acid/base catalysts.

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Section: Desolvation -Activate the Nucleophile And The Electrophilesupporting

confidence: 82%

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

et al. 2017

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“…Noteworthy is also the development of enzymatic ligation methods for the preparation of proteins with enzymes specifically engineered to perform reverse proteolysis and to act as "ligases". [17] Nevertheless, despite some notable successes, [18][19] such methods have not found widespread use (yet) after the development of the more simple and versatile chemical ligation methods. [1,[20][21][22][23][24] The size of proteins which can be chemically synthesized has been increased Because the mutually reactive groups (or at least one of them) are functionalities not normally found in peptides, the price to be paid for applying such chemoselective ligation is the formation of an unnatural structure at the ligation site.…”

Section: Chemical Protein Synthesismentioning

confidence: 99%

Diels–Alder Ligation of Peptides and Proteins

Araújo

Palomo

Cramer

et al. 2006

Chemistry A European J

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“…To date, there are just a few examples of designed proteins that adopt a specified fold, and number which display novel activities is still smaller (Dill, 1990; Schafmeister et al, 1993;Jackson et al, 1994;Quinn et al, 1994;Yan & Erickson, 1994;Baltzer et al, 1996;Dahiyat & Mayo, 1997;Hill & DeGrado, 1998;QuCnCneur et al, 1998). A particular focus of functional design efforts has been the engineering of novel metal-binding sites.…”

Section: Introductionmentioning

confidence: 99%

The de novo design of a rubredoxin‐like fe site

Farinas

Regan

1998

Protein Science

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A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B 1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [ F e ( S -c~s )~] ",-'. The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(I1) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(I1)-bound forms of the variant are folded correctly and are stable. The Fe(II1) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(II1) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.Keywords: de novo design; metalloprotein; protein engineering; rubredoxin Protein engineering and de novo design approaches have begun to afford a more detailed understanding of the balance of the forces that determine protein structure and function (Regan & DeGrado, 1988;Regan, 1993;Bryson et al., 1995;Regan, 1995;Dalal et al., 1997;Smith & Regan, 1997;Hellinga, 1998). To date, there are just a few examples of designed proteins that adopt a specified fold, and number which display novel activities is still smaller (Dill, 1990; Schafmeister et al., 1993;Jackson et al., 1994;Quinn et al., 1994;Yan & Erickson, 1994;Baltzer et al., 1996;Dahiyat & Mayo, 1997;Hill & DeGrado, 1998;QuCnCneur et al., 1998). A particular focus of functional design efforts has been the engineering of novel metal-binding sites. Metal-binding sites represent an attractive target because they play a variety of functions from stabilization of protein structure to catalytic roles such as the activation of water for nucleophilic attack, electron transfer, and redox activity. More recently, several groups have focused strongly on the design of metal-binding sites with particular activities in mind. For example, two metal-binding histidine residues have been engineered into rat trypsin to "switch" the catalytic activity on or off by disrupting the catalytic triad. The protein can be turned off by Cu(I1) chelation in the designed site and reactivated upon removal of Cu(I1) with EDTA (Halfon & Craik, 1996). In another design, a metal-binding site, based on the metal center of carbonic

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A Designed Peptide Ligase for Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues

Cited by 297 publications

References 35 publications

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

Diels–Alder Ligation of Peptides and Proteins

The de novo design of a rubredoxin‐like fe site

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