Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

Weinstock, Matthew T.; Jacobsen, Michael T.; Kay, Michael S.

doi:10.1073/pnas.1410900111

Cited by 131 publications

(90 citation statements)

References 55 publications

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“…55 Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression, including improvement of mRNA stability and translational efficiency, protein folding by chaperone co-expression, expression of disulfide-bonded proteins, acetylated and glycosylated protein production have been developed. 51,61,62 Strains that confer improved protein expression can be engineered by screening libraries of chromosomal mutants as well as plasmid-encoded expression libraries of heterologous or native genes. 51,58,59,60 The directed evolution based approach to increase protein production levels by the vector backbone optimization has also been reported.…”

Section: Genetic Modification Of Producing Strainsmentioning

confidence: 99%

Genetically modified proteins: functional improvement and chimeragenesis

et al. 2015

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This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit.

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Section: Genetic Modification Of Producing Strainsmentioning

confidence: 99%

Genetically modified proteins: functional improvement and chimeragenesis

et al. 2015

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“…D peptides | transmembrane peptides | racemic crystallization | racemic detergent | coiled coil P olypeptides comprising D-amino acid residues have been sources of growing interest for biological applications, often for functions that depend on recognition by specific natural proteins (1)(2)(3). D peptides offer identical versatility in terms of conformation and side-chain functionality relative to conventional peptides (composed of L-amino acid residues), but D peptides are impervious to the action of proteolytic enzymes, which should improve pharmacokinetic properties in vivo relative to those of conventional peptides.…”

mentioning

confidence: 99%

High-resolution structures of a heterochiral coiled coil

Mortenson

Steinkruger

Kreitler

et al. 2015

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

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Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from D amino acids (D peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious proteinprotein interactions. Coiled-coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled-coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between L-and D-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.D peptides | transmembrane peptides | racemic crystallization | racemic detergent | coiled coil P olypeptides comprising D-amino acid residues have been sources of growing interest for biological applications, often for functions that depend on recognition by specific natural proteins (1-3). D peptides offer identical versatility in terms of conformation and side-chain functionality relative to conventional peptides (composed of L-amino acid residues), but D peptides are impervious to the action of proteolytic enzymes, which should improve pharmacokinetic properties in vivo relative to those of conventional peptides. The engineering of D peptides to display defined protein-binding preferences is hindered, however, by the dearth of experimental information available for such complexes. Structural principles that are well-known to govern interactions between two L-polypeptide chains are not directly extensible to pairings between peptides of opposite chirality. Favorable heterochiral interactions (between L-and D peptides) that are analogous to homochiral associations between L peptides were postulated decades ago on the basis of geometrical considerations (4, 5). In a 1953 analysis of structural parameters governing coiled-coil formation between right-handed α-helices formed from L peptides, for example, Crick suggested that analogous assemblies should be accessible to pairs of right-and left-handed helices (4). In the same year, Pauling and Corey postulated that heterochiral peptide mixtures could form "rippled" β-sheet assemblies with backbo...

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“…Solubility : In our experience 46,59,76,77 , the handling properties of peptides under NCL and acidic RP-HPLC conditions correlate positively with the density of positively charged residues (His, Lys, Arg) and negatively with the density of negatively charged or branched hydrophobic residues (Asp, Glu, Val, Ile, and Leu). Therefore, we assign a score of +1 for each HKR residue and −1 for each DEVIL residue.…”

Section: Discussionmentioning

confidence: 82%

“…2A):

Thioester (ligation) kinetics 73 : Segments containing a preferred C-terminal thioester receive +2 points (Ala, Arg, Cys, Gly, His, Met, Phe, Ser, Trp, or Tyr), while other acceptable thioesters (Ile, Leu, Lys 75 , Thr, or Val) result in 0 points. See File S3 for details.

Solubility : In our experience 46,59,76,77 , the handling properties of peptides under NCL and acidic RP-HPLC conditions correlate positively with the density of positively charged residues (His, Lys, Arg) and negatively with the density of negatively charged or branched hydrophobic residues (Asp, Glu, Val, Ile, and Leu). Therefore, we assign a score of +1 for each HKR residue and −1 for each DEVIL residue.…”

Section: Discussionmentioning

confidence: 83%

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Aligator: A computational tool for optimizing total chemical synthesis of large proteins

Jacobsen

Erickson

Kay

2017

Bioorganic & Medicinal Chemistry

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The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.

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Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

Cited by 131 publications

References 55 publications

Genetically modified proteins: functional improvement and chimeragenesis

Genetically modified proteins: functional improvement and chimeragenesis

High-resolution structures of a heterochiral coiled coil

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

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