Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent
The de novo design of proteins that bind highly functionalized small molecules represents a great challenge. To enable computational design of binders, we developed a unit of protein structure—a van der Mer (vdM)—that maps the backbone of each amino acid to statistically preferred positions of interacting chemical groups. Using vdMs, we designed six de novo proteins to bind the drug apixaban; two bound with low and submicromolar affinity. X-ray crystallography and mutagenesis confirmed a structure with a precisely designed cavity that forms favorable interactions in the drug–protein complex. vdMs may enable design of functional proteins for applications in sensing, medicine, and catalysis.
more » « less- Award ID(s):
- 1709506
- NSF-PAR ID:
- 10190463
- Publisher / Repository:
- American Association for the Advancement of Science (AAAS)
- Date Published:
- Journal Name:
- Science
- Volume:
- 369
- Issue:
- 6508
- ISSN:
- 0036-8075
- Page Range / eLocation ID:
- p. 1227-1233
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract De novo design provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron–sulfur clusters, the design of tetranuclear clusters with oxygen‐rich environments remains in its infancy. In previous work, we described the design of homotetrameric four‐helix bundles that bind tetra‐Zn2+clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal‐binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the correspondingapo ‐proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn2+in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in theapo state, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster. -
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Abstract Extensive efforts invested in understanding the rules of protein folding are now being applied, with good effect, in de novo design of proteins/peptides. For proteins containing standard α‐amino acids alone, knowledge derived from experimentally determined three‐dimensional (3D) structures of proteins and biologically active peptides are available from the Protein Data Bank (PDB), and the Cambridge Structural Database (CSD). These help predict and design protein structures, with reasonable confidence. However, our knowledge of 3D structures of biomolecules containing backbone modified amino acids is still evolving. A major challenge in de novo protein/peptide design concerns the engineering of conformationally constrained molecules with specific structural elements and chemical groups appropriately positioned for biological activity. This review explores four classes of amino acid modifications that constrain protein/peptide backbone structure. Systematic analysis of peptidic molecule structures (eg, bioactive peptides, inhibitors, antibiotics, and designed molecules), containing these backbone‐modified amino acids, found in the PDB and CSD are discussed. The review aims to provide structure–function insights that will guide future design of proteins/peptides.
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Abstract While native proteins cover diverse structural spaces and achieve various biological events, not many of them can directly serve human needs. One reason is that the native proteins usually contain idiosyncrasies evolved for their native functions but disfavoring engineering requirements. To overcome this issue, one strategy is to create de novo proteins which are designed to possess improved stability, high environmental tolerance, and enhanced engineering potential. Compared to other protein engineering strategies, in silico design of de novo proteins has significantly expanded the protein structural and sequence spaces, reduced wet lab workload, and incorporated engineered features in a guided and efficient manner. In the Baker laboratory we have been applying a design pipeline that uses the blueprint builder to design different folds of de novo proteins, and have successfully obtained libraries of de novo proteins with improved stability and engineering potential. In this article, we will use the design of de novo β‐barrel proteins as an example to describe the principles and basic procedures of the blueprint builder−based design pipeline. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : The construction of blueprintsAlternate Protocol : Build blueprints based on existing protein.pdb filesBasic Protocol 2 : De novo protein design pipeline using the blueprint builder
