A Strategy for Combinatorial Cavity Design in De Novo Proteins

Abstract

Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there solutions to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To investigate these questions, we have created combinatorial collections, or libraries, of novel sequences with no homology to those found in living organisms. Previously designed libraries contained numerous functional proteins. However, they often formed dynamic, rather than well-ordered structures, which complicated structural and mechanistic characterization. To address this challenge, we describe the development of new libraries based on the de novo protein S-824, a 4-helix bundle with a very stable 3-dimensional structure. Distinct from previous libraries, we targeted variability to a specific region of the protein, seeking to create potential functional sites. By characterizing variant proteins from this library, we demonstrate that the S-824 scaffold tolerates diverse amino acid substitutions in a putative cavity, including buried polar residues suitable for catalysis. We designed and created a DNA library encoding 1.7 × 106 unique protein sequences. This new library of stable de novo α-helical proteins is well suited for screens and selections for a range of functional activities in vitro and in vivo.

Root of the Tree: The Significance, Evolution, and Origins of the Ribosome

“…The universe lacks sufficient atoms or time to accomplish a random search of even a small subspace of this landscape. 279,280 Instead, the rare suite of features that conferred folding and assembly competence to Nature's functional biopolymers was obtained via repeated exaptation processes. The evolution of proteins was hierarchical and progressive.…”

“…The vast space of possible monomer-types, backbone chemistries, side chain types, polymer topologies, and sequences was not fully explored during the evolution of proteins. The universe lacks sufficient atoms or time to accomplish a random search of even a small subspace of this landscape. , Instead, the rare suite of features that conferred folding and assembly competence to Nature’s functional biopolymers was obtained via repeated exaptation processes. The evolution of proteins was hierarchical and progressive.…”

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“…It was extended in a procedure dubbed ISOR (Incorporating Synthetic Oligonucleotides via Gene Re‐assembly) . Synthetic DNA shuffling was also developed and applied in directed evolution, e.g., Assembly of Designed Oligonucleotides (ADO) . The problem of low homology in shuffling was addressed in a number of other studies, reviewed in 2016 .…”

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“…As an example of how variable libraries can be used to develop cavities and small molecule binding sites, Karas and Hecht developed libraries based on the de novo designed 4-helix bundle protein S-824 [ 45 ]. The library created by the authors consists of 1.7 × 10 6 unique sequences.…”