High thermodynamic stability of parametrically designed helical bundles

Huang, Po-Ssu; Oberdorfer, Gustav; Xu, Chunfu; Pei, X.Y.; Nannenga, Brent L.; Rogers, Joseph M.; DiMaio, Frank; Gonen, Tamir; Luisi, Ben F.; Baker, David

doi:10.1126/science.1257481

Cited by 269 publications

(307 citation statements)

References 35 publications

(23 reference statements)

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“…It has previously been suggested that heterochiral α-helical interfaces can occur over only about four heptad repeats before core side chains diverge from the interhelical interface; however, we speculate that longer and likely more stable interfaces could be formed between L-and D polypeptides containing nonheptad sequence repeats. Recent work has shown that native and designed L polypeptides harboring a hendecad sequence pattern, either alone or in combination with the heptad repeat, can form homochiral quaternary assemblies with little or no supercoiling (50,53). This trend is significant in the context of heterochiral assemblies because oligomers formed between L-and D polypeptides are expected to be incompatible with α-helical supercoiling.…”

Section: Discussionmentioning

confidence: 97%

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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Section: Discussionmentioning

confidence: 97%

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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“…However, we posited that, in contrast to most natural protein structures, the high stability of de novo -helical barrels and bundles might tolerate this level of change. 35,40 To test this, we adopted an iterative design strategy, fully characterizing point and double mutants en route. …”

Section: Rational Design Of Multiple Active Sitesmentioning

confidence: 99%

Installing hydrolytic activity into a completely de novo protein framework

et al. 2016

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Designing enzyme-like catalysts tests our understanding of sequence-tostructure/function relationships in proteins. Here, we install hydrolytic activity predictably into a completely de novo and thermo-stable -helical barrel, which comprises 7 helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses pnitrophenyl acetate with catalytic efficiencies matching the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.(134 words)

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“…We demonstrate a high level of design success for ThreeFoil as evidenced by its: (i) reversible, cooperative, two-state (un)folding; and (ii) well folded and functional native structure which has high solubility and monodispersity, well diffracting crystals, and great resistance against H/D exchange (1), denaturation by chaotropes and detergent, and degradation by protease. Although the rational design of proteins with desired structure and function remains a great challenge and often require multiple cycles of design and/or selection to improve them, successes in designing both structures and/or functions, including ones not observed in nature, have been increasing (3,4,6,8,9,18,49,50). These results demonstrate the increasing understanding of fundamental principles and utility of computational protein design.…”

Section: High Chemical and Protease Resistances Of Threefoil And Othementioning

confidence: 83%

Designed protein reveals structural determinants of extreme kinetic stability

Broom

Xia

et al. 2015

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

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The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/ LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge.SDS/protease resistance | protein folding | coarse-grained simulations | protein topology | contact order

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High thermodynamic stability of parametrically designed helical bundles

Cited by 269 publications

References 35 publications

High-resolution structures of a heterochiral coiled coil

High-resolution structures of a heterochiral coiled coil

Installing hydrolytic activity into a completely de novo protein framework

Designed protein reveals structural determinants of extreme kinetic stability

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