A protein constructed de novo enables cell growth by altering gene regulation

Abstract

Recent advances in protein design rely on rational and computational approaches to create novel sequences that fold and function. In contrast, natural systems selected functional proteins without any design a priori. In an attempt to mimic nature, we used large libraries of novel sequences and selected for functional proteins that rescue Escherichia coli cells in which a conditionally essential gene has been deleted. In this way, the de novo protein SynSerB3 was selected as a rescuer of cells in which serB, which encodes phosphoserine phosphatase, an enzyme essential for serine biosynthesis, was deleted. However, SynSerB3 does not rescue the deleted activity by catalyzing hydrolysis of phosphoserine. Instead, SynSerB3 upregulates hisB, a gene encoding histidinol phosphate phosphatase. This endogenous E. coli phosphatase has promiscuous activity that, when overexpressed, compensates for the deletion of phosphoserine phosphatase. Thus, the de novo protein SynSerB3 rescues the deletion of serB by altering the natural regulation of the His operon.O ne of the key goals of synthetic biology is to design novel proteins that fold and function in vivo. A particularly challenging objective would be to produce nonnatural proteins that do not merely generate interesting phenotypes but actually provide essential functions necessary for the growth of living cells. The successful design of such life-sustaining proteins would represent an initial step toward constructing artificial "proteomes" of nonnatural sequences.We initiated work toward artificial proteomes by constructing large combinatorial libraries of novel sequences designed to fold into stable four-helix structures (1, 2). Our libraries were based on a strategy for protein design, which assumes that the overall fold of a simple structure can be specified by the pattern of polar and nonpolar residues in the linear sequence. Because only the type of residue-polar versus nonpolar-is specified, this strategy has been called a "binary code" for protein design (1-3). At the same time, because the exact identities of the side chains at each polar and nonpolar position are not specified explicitly, this strategy is well suited for constructing large combinatorial libraries of novel sequences (3). To express these libraries of binary-patterned sequences in vivo, we construct collections of synthetic genes using degenerate DNA codons. For example, the degenerate codon NTN (N = A,T,C,G) is used to encode five nonpolar residues, and the degenerate codon VAN (V = A,C,G) is used to encode six polar residues.We have shown previously that several proteins from these binary patterned libraries fold into stable four-helix bundles, and both crystallographic and NMR structures have been determined (4-6). Moreover, in initial steps probing the potential for functional activity, proteins from these libraries were shown to bind small molecules, including cofactors, and to catalyze rudimentary reactions (7,8). Although those experiments screened a subset of library proteins for activity in ...

Structural and Functional Modeling of Artificial Bioactive Proteins

“…Screening of the third-generation computational libraries for de novo sequences that function in vivo yielded several Hecht_α sequences, termed SynRescue proteins [3,6], that rescue conditionally lethal mutants of Escherichia coli (auxotrophs) [3,6,11]. From a practical standpoint, it would be desirable not only to construct and test a large number of structural patterns in proteins, but also to predict the functional characteristics of the newly-designed molecules.…”

“…However, there are several sets of well-characterized artificial proteins that may be used for the testing of algorithms concerning protein structure and function [1][2][3][4][5][6]. In our research, we tested standard bioinformatics methods using the de novo protein subset of Hecht et al [3,6,[8][9][10][11][12]. It represents a well-characterized and sufficiently large subset of 15 synthetic α-and 17 β-proteins (Hecht_α and Hecht_β) [3,6,[8][9][10][11][12][13].…”

“…In our research, we tested standard bioinformatics methods using the de novo protein subset of Hecht et al [3,6,[8][9][10][11][12]. It represents a well-characterized and sufficiently large subset of 15 synthetic α-and 17 β-proteins (Hecht_α and Hecht_β) [3,6,[8][9][10][11][12][13]. A dataset of this size was sufficient for this study due to the small number of variables analyzed in comparison to the number of members of each class (Hecht_α and Hecht_β) [14].…”

“…They showed that complex molecular structures could be made using amino acid polarity patterns pnppnnp and pnpnpnp that define stable α-helices and β-strands, respectively [8][9][10]. Hecht_α proteins were not explicitly designed to be functional, but recent studies have shown that they also provide the biological functions necessary to maintain cell growth where genes encoding enzymes essential for amino acid biosynthesis have been deleted [6,11]. They have been named SynRescue proteins [3,6,11].…”

“…Hecht_α proteins were not explicitly designed to be functional, but recent studies have shown that they also provide the biological functions necessary to maintain cell growth where genes encoding enzymes essential for amino acid biosynthesis have been deleted [6,11]. They have been named SynRescue proteins [3,6,11].…”