De Novo Design of a Single-Chain Diphenylporphyrin Metalloprotein

Bender, Gretchen M.; Lehmann, Andreas; Zou, Hongling; Cheng, Hong; Fry, H. Christopher; Engel, Don; Therien, Michael J.; Blasie, J. Kent; Röder, Heinrich; Saven, Jeffrey G.; DeGrado, William F.

doi:10.1021/ja071199j

Cited by 91 publications

(99 citation statements)

References 37 publications

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“…R and θ values, where the backbone atoms overlap, were discounted. In the context of a protein's nearest neighbors within each crystalline configuration, the site-specific probabilities of the 18 allowed amino acids, their side-chain conformations, and the weighted average energy over these sequence-rotamer states EðR; θÞ were estimated computationally (30,31,47). Effective solvation energy functions were not used (31), and no constraint was placed on the net charge of the protein.…”

Section: Resultsmentioning

confidence: 99%

Computational design of a protein crystal

Lanci

MacDermaid

Kang

et al. 2012

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

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157

165

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Protein crystals have catalytic and materials applications and are central to efforts in structural biology and therapeutic development. Designing predetermined crystal structures can be subtle given the complexity of proteins and the noncovalent interactions that govern crystallization. De novo protein design provides an approach to engineer highly complex nanoscale molecular structures, and often the positions of atoms can be programmed with sub-Å precision. Herein, a computational approach is presented for the design of proteins that self-assemble in three dimensions to yield macroscopic crystals. A three-helix coiled-coil protein is designed de novo to form a polar, layered, three-dimensional crystal having the P6 space group, which has a "honeycomb-like" structure and hexameric channels that span the crystal. The approach involves: (i) creating an ensemble of crystalline structures consistent with the targeted symmetry; (ii) characterizing this ensemble to identify "designable" structures from minima in the sequencestructure energy landscape and designing sequences for these structures; (iii) experimentally characterizing candidate proteins. A 2.1 Å resolution X-ray crystal structure of one such designed protein exhibits sub-Å agreement [backbone root mean square deviation (rmsd)] with the computational model of the crystal. This approach to crystal design has potential applications to the de novo design of nanostructured materials and to the modification of natural proteins to facilitate X-ray crystallographic analysis.M olecular design provides powerful tools for exploring how molecular properties dictate macroscopic structure and function. One of the most precise forms of self-organization, crystallization achieves orientation and symmetry across many length scales and can be leveraged to engineer materials with well-defined molecular order (1). In addition to their central role in structure determination, molecular crystals have many applications, including nanoparticle templating (2, 3), nonlinear optical devices (4), molecular scaffolding (5), and porous frameworks (6). A predictive understanding of how to achieve self-assembled macroscale structure and desired properties remains challenging, however, particularly when large, conformationally flexible molecules are employed.Small synthetic molecules have been designed that display complementary functional groups in a manner consistent with a chosen crystal structure. Intermolecular contacts are often patterned using strong, directional interactions (e.g., hydrogen bonding, metalcoordination, and electrostatic interactions) (7). The constituent molecules, however, are typically small and synthetic, thus limiting size, shape, and functionality. Hence, simultaneously achieving functional properties and the presentation of complementary intermolecular interactions that confer a targeted crystal structure can be difficult. Commonly, weak intermolecular forces stabilize crystalline ordering, and as a result, quantitative, predictive approaches to crystal de...

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Section: Resultsmentioning

confidence: 99%

Computational design of a protein crystal

Lanci

MacDermaid

Kang

et al. 2012

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

Self Cite

157

165

View full text Add to dashboard Cite

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“…This is illustrated by the backbone shifts that accompany functional divergence in natural protein evolution. Previous studies have achieved functional changes by backbone alteration, but relied on grafting methods that are restricted to sequences of known structure and function (8)(9)(10), which may be suboptimal with respect to the desired property.…”

mentioning

confidence: 99%

Alteration of enzyme specificity by computational loop remodeling and design

Murphy

Bolduc

Gallaher

et al. 2009

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

113

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Altering the specificity of an enzyme requires precise positioning of side-chain functional groups that interact with the modified groups of the new substrate. This requires not only sequence changes that introduce the new functional groups but also sequence changes that remodel the structure of the protein backbone so that the functional groups are properly positioned. We describe a computational design method for introducing specific enzyme-substrate interactions by directed remodeling of loops near the active site. Benchmark tests on 8 native protein-ligand complexes show that the method can recover native loop lengths and, often, native loop conformations. We then use the method to redesign a critical loop in human guanine deaminase such that a key side-chain interaction is made with the substrate ammelide. The redesigned enzyme is 100-fold more active on ammelide and 2.5e4-fold less active on guanine than wild-type enzyme: The net change in specificity is 2.5e6-fold. The structure of the designed protein was confirmed by X-ray crystallographic analysis: The remodeled loop adopts a conformation that is within 1-Å C␣ RMSD of the computational model. computational protein design ͉ loop modeling C omputational protein design methodology has been used to optimize properties such as protein stability (1, 2) and to introduce functions such as binding of small molecules (3), proteins (4), and nucleic acids (5), as well as enzymatic activity (6, 7). In most of these studies, the implicit assumption that the structure of the polypeptide backbone would remain largely fixed despite mutations of amino acid side chains was made for the sake of computational tractability.Explicit remodeling of the polypeptide backbone makes possible further optimization of these and other structural or functional properties. The set of combinations of protein sequences and structures is considerably larger when backbone flexibility is allowed and is likely to contain conformations that optimize a desired property to a greater degree than the original scaffold. This is illustrated by the backbone shifts that accompany functional divergence in natural protein evolution. Previous studies have achieved functional changes by backbone alteration, but relied on grafting methods that are restricted to sequences of known structure and function (8-10), which may be suboptimal with respect to the desired property.De novo protein structure prediction methods are well suited for sampling novel backbone conformations (11). These methods have recently been extended to focus sampling on conformations that satisfy specific positional constraints (12, 13). Computational design algorithms that iterate between sequence design and backbone optimization using structure-prediction methods have been used to design previously unobserved protein fold and loop conformations (2, 14), but have not yet been applied to achieving functional changes such as alteration of an enzyme's substrate specificity.We have developed a computational design algorithm that uses constr...

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“…For this reason we connected two copies of the HP7 gene to each other, creating the single chain four helix bundle protein H4 (see Figure 1C). This strategy was used by Bender and coworkers to make a single chain four alpha helix bundle which binds pairs of diphenyl porphyrins (Bender et al, 2007). In order to ease future engineering efforts, each loop was constructed to contain a unique restriction site at the DNA level, which results in minor variation in each loop sequence over the original glycine and serine loops.…”

Section: Resultsmentioning

confidence: 99%

Rational design of a zinc phthalocyanine binding protein

Mutter¹,

Norman²,

Tiedemann³

et al. 2014

Journal of Structural Biology

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Phthalocyanines have long been used as primary donor molecules in synthetic light-powered devices due to their superior properties when compared to natural light activated molecules such as chlorophylls. Their use in biological contexts, however, has been severely restricted due to their high degree of self-association, and its attendant photoquenching, in aqueous environments. To this end we report the rational redesign of a de novo four helix bundle di-heme binding protein into a heme and Zinc(II) phthalocyanine (ZnPc) dyad in which the ZnPc is electronically and photonically isolated. The redesign required transformation of the homodimeric protein into a single chain four helix bundle and the addition of a negatively charge sulfonate ion to the ZnPc macrocycle. To explore the role of topology on ZnPc binding two constructs were made and the resulting differences in affinity can be explained by steric interference of the newly added connecting loop. Singular binding of ZnPc was verified by absorption, fluorescence, and magnetic circular dichroism spectroscopy. The engineering guidelines determined here, which enable the simple insertion of a monomeric ZnPc binding site into an artificial helical bundle, are a robust starting point for the creation of functional photoactive nanodevices.

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De Novo Design of a Single-Chain Diphenylporphyrin Metalloprotein

Cited by 91 publications

References 37 publications

Computational design of a protein crystal

Computational design of a protein crystal

Alteration of enzyme specificity by computational loop remodeling and design

Rational design of a zinc phthalocyanine binding protein

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