Binding of small molecules to cavity forming mutants of a <i>de novo</i> designed protein

Das, Aditi; Wei, Yinan; Pelczer, István; Hecht, Michael H.

doi:10.1002/pro.601

Cited by 9 publications

(8 citation statements)

References 61 publications

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“…Two relatively large pockets (volumes: 205 and 174 Å 3 ), comprised of Leu30, Met33, Asn34, Met37, His74, and Leu75 (in chain A/B) and Leu16, Leu19, Phe85, Leu89, and Leu92 (in chain B/A) are detected using the programs Pocket-Finder () and Cavor () (Figure ). Similar sized pockets were also found in the F64A mutant of monomeric protein S-824 . We hypothesize that these hydrophobic pockets may serve as substrate binding sites.…”

Section: Resultssupporting

confidence: 68%

Domain-Swapped Dimeric Structure of a Stable and Functional De Novo Four-Helix Bundle Protein, WA20

et al. 2012

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To probe the potential for activity in unevolved amino acid sequence space, we created a third generation combinatorial library of de novo four-helix bundle proteins. The "artificial superfamily" of helical bundles was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. WA20, picked from the library, is one of the most stable proteins in the superfamily, and has rudimentary activities such as esterase and lipase. Here we report the crystal structure of WA20, determined by the multiwavelength anomalous dispersion method. Unexpectedly, the WA20 crystal structure is not a monomeric four-helix bundle, but a dimeric four-helix bundle. Each monomer comprises two long α-helices that intertwist with the helices of the other monomer. The two monomers together form a 3D domain-swapped four-helix bundle dimer. In addition, there are two hydrophobic pockets, which may potentially provide substrate binding sites. Small-angle X-ray scattering shows that the molecular weight of WA20 is ~25 kDa and the shape is rod-like (the maximum length, D(max) = ~8 nm), indicating that WA20 forms a dimeric four-helix bundle in solution. These results demonstrate that our de novo protein library contains not only simple monomeric proteins, but also stable and functional multimeric proteins.

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Section: Resultssupporting

confidence: 68%

Domain-Swapped Dimeric Structure of a Stable and Functional De Novo Four-Helix Bundle Protein, WA20

et al. 2012

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“…To confirm binding and estimate affinities, we used pulse field gradient spin echo (PFGSE) NMR (21). This method is well-suited to study the affinities between macromolecules and SMs, and we have used it previously to study binding to novel proteins (22). PFGSE NMR enables estimation of binding affinities from the mole fraction of the protein/ligand complex, which is estimated from the observed diffusion coefficients of the ligand in the free and bound states.…”

Section: Resultsmentioning

confidence: 99%

“…21 This method is well-suited to study the affinities between macromolecules and SMs, and we have used it previously to study binding to novel proteins. 22 PFGSE NMR enables estimation of binding affinities from the mole fraction of the protein/ligand complex, which is estimated from the observed diffusion coefficients of the ligand in the free and bound states. PFGSE experiments monitoring the interaction of paromomycin with S836 and S23 yielded K D values in the range of 15−20 μM (Figure 4a).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Proteins from an Unevolved Library of de novo Designed Sequences Bind a Range of Small Molecules

et al. 2012

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The availability of large collections of de novo designed proteins presents new opportunities to harness novel macromolecules for synthetic biological functions. Many of these new functions will require binding to small molecules. Is the ability to bind small molecules a property that arises only in response to biological selection or computational design? Or alternatively, is small molecule binding a property of folded proteins that occurs readily amidst collections of unevolved sequences? These questions can be addressed by assessing the binding potential of de novo proteins that are designed to fold into stable structures, but are “naïve” in the sense that they (i) share no significant sequence similarity with natural proteins, and (ii) were neither selected nor designed to bind small molecules. We chose three naïve proteins from a library of sequences designed to fold into 4-helix bundles, and screened for binding to 10,000 compounds displayed on small molecule microarrays. Several binders were identified, and binding was characterized by a series of biophysical assays. Surprisingly, despite the similarity of the three de novo proteins to one another, they exhibit selective ligand binding. These findings demonstrate the potential of novel proteins for molecular recognition, and have significant implications for a range of applications in synthetic biology.

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“…We have used the binary code strategy to design several libraries of α‐helical and β‐sheet proteins, and have demonstrated that proteins from the α‐helical libraries fold into well‐ordered stable structures . Proteins from these libraries bind small molecules, including cofactors, and catalyze reactions . Most importantly, several of these de novo α‐helical proteins function in vivo to provide essential activities necessary to sustain the growth of living cells …”

Section: Introductionmentioning

confidence: 99%

A de novo protein confers copper resistance in Escherichia coli

Hoegler

Hecht

2016

Protein Science

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To survive environmental challenges, biological systems rely on proteins that were selected by evolution to function in particular cellular and conditional settings. With the advent of protein design and synthetic biology, it is now possible to construct novel proteins that are not biased by eons of selection in natural hosts. The availability of these sequences prompts us to ask whether natural biological organisms can use na€ ıve-non-biological-proteins to enhance fitness in stressful environments. To address this question, we transformed a library of DNA sequences encoding~1.5 3 10 6 binary patterned de novo proteins into E. coli, and selected for sequences that enable growth in concentrations of copper that would otherwise be toxic. Several novel sequences were discovered, and one of them, called Construct K (ConK), was studied in detail. Cells expressing ConK accumulate approximately 50% less copper than control cells. The function of ConK does not involve an oxidase, nor does it require two of the best characterized copper efflux systems. However, the ability of ConK to rescue cells from toxic concentrations of copper does require an active proton motive force. Further selections for growth in higher concentrations of copper led to the laboratory evolution of variants of ConK with enhanced levels of activity in vivo. These studies demonstrate that novel proteins, unbiased by evolutionary history in the natural world, can enhance the fitness of biological systems.Synopsis: Living systems evolve to adapt to potentially lethal environmental changes. This normally involves repurposing existing genetic information (i.e. sequences that were selected by billions of years of evolution). Here we show that a completely de novo protein, not derived from nature, can enable E. coli cells to grow in otherwise toxic concentrations of copper, demonstrating that living systems also have the capacity to incorporate and protopurpose entirely novel genetic information.

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Binding of small molecules to cavity forming mutants of a de novo designed protein

Cited by 9 publications

References 61 publications

Domain-Swapped Dimeric Structure of a Stable and Functional De Novo Four-Helix Bundle Protein, WA20

Domain-Swapped Dimeric Structure of a Stable and Functional De Novo Four-Helix Bundle Protein, WA20

Proteins from an Unevolved Library of de novo Designed Sequences Bind a Range of Small Molecules

A de novo protein confers copper resistance in Escherichia coli

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