De Novo Protein Design: Fully Automated Sequence Selection

Dahiyat, Bassil I.; Mayo, Stephen L.

doi:10.1126/science.278.5335.82

Cited by 1,074 publications

(967 citation statements)

References 41 publications

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“…The requirement to not bury lone charges would be a reasonable mechanism for reducing structural degeneracy (Paul, 1982). This idea leads to the design principle of plac- ing hydrophobic groups at buried positions and hydrophilic ones at exposed positions (Kametekar et al, 1993;Munson et al, 1994;Dahiyat & Mayo, 1997). An analysis was performed to examine the question of whether lone charges or salt bridging charges played a larger role in reducing structural degeneracy.…”

Section: Resultsmentioning

confidence: 99%

Effects of salt bridges on protein structure and design

Sindelar¹,

Hendsch²,

Tidor

1998

Protein Science

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Theoretical calculations (Hendsch ZS & Tidor B, 1994, Protein Sci 3:211-226) and experiments (Waldburger CD et al., 1995, Nut Struct Biol 2:122-128;Wimley WC et al., 1996, Proc Natl Acud Sci USA 93:2985-2990) suggest that hydrophobic interactions are more stabilizing than salt bridges in protein folding. The lack of apparent stability benefit for many salt bridges requires an alternative explanation for their occurrence within proteins. To examine the effect of salt bridges on protein structure and stability in more detail, we have developed an energy function for simple cubic lattice polymers based on continuum electrostatic calculations of a representative selection of salt bridges found in known protein crystal structures. There are only three types of residues in the model, with charges of -1, 0, or + 1. We have exhaustively enumerated conformational space and significant regions of sequence space for three-dimensional cubic lattice polymers of length 16. The results demonstrate that, while the more highly charged sequences are less stable, the loss of stability is accompanied by a substantial reduction in the degeneracy of the lowest-energy state. Moreover, the reduction in degeneracy is greater due to charges that pair than for lone charges that remain relatively exposed to solvent. We have also explored and illustrated the use of ion-pairing strategies for rational structural design using model lattice studies.

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Section: Resultsmentioning

confidence: 99%

Effects of salt bridges on protein structure and design

Sindelar¹,

Hendsch²,

Tidor

1998

Protein Science

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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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“…Computational protein design methods can identify de novo amino acid sequences that can be folded into predefined topologies. 156,157 As a representative example, the 28-residue full sequence design (FSD-1) bba-protein was computationally designed by Dahiyat and Mayo to form a stable zinc-finger bba-fold independent of zinc binding. 156 Starting with the backbone coordinates of the zinc-finger protein Zif268, they selected side-chain rotamers to optimize side-chain/side-chain and backbone/side-chain interactions.…”

Section: Toward Protein Engineeringmentioning

confidence: 99%

“…156,157 As a representative example, the 28-residue full sequence design (FSD-1) bba-protein was computationally designed by Dahiyat and Mayo to form a stable zinc-finger bba-fold independent of zinc binding. 156 Starting with the backbone coordinates of the zinc-finger protein Zif268, they selected side-chain rotamers to optimize side-chain/side-chain and backbone/side-chain interactions. The designed protein was synthesized and its structure solved by NMR; the resulting structure's overall backbone RMSD was 1.98 Å relative to the computationally designed target.…”

Section: Toward Protein Engineeringmentioning

confidence: 99%

Back to the future: Ribonuclease A

2008

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Pancreatic ribonuclease A (EC 3.1.27.5, RNase) is, perhaps, the best‐studied enzyme of the 20th century. It was isolated by René Dubos, crystallized by Moses Kunitz, sequenced by Stanford Moore and William Stein, and synthesized in the laboratory of Bruce Merrifield, all at the Rockefeller Institute/University. It has proven to be an excellent model system for many different types of experiments, both as an enzyme and as a well‐characterized protein for biophysical studies. Of major significance was the demonstration by Chris Anfinsen at NIH that the primary sequence of RNase encoded the three‐dimensional structure of the enzyme. Many other prominent protein chemists/enzymologists have utilized RNase as a dominant theme in their research. In this review, the history of RNase and its offspring, RNase S (S‐protein/S‐peptide), will be considered, especially the work in the Merrifield group, as a preface to preliminary data and proposed experiments addressing topics of current interest. These include entropy–enthalpy compensation, entropy of ligand binding, the impact of protein modification on thermal stability, and the role of protein dynamics in enzyme action. In continuing to use RNase as a prototypical enzyme, we stand on the shoulders of the giants of protein chemistry to survey the future. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 259–277, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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De Novo Protein Design: Fully Automated Sequence Selection

Cited by 1,074 publications

References 41 publications

Effects of salt bridges on protein structure and design

Effects of salt bridges on protein structure and design

The de novo design of a rubredoxin‐like fe site

Back to the future: Ribonuclease A

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