title: De Novo Proteins with Life-Sustaining Functions Are Structurally Dynamic.
abstract: Designing and producing novel proteins that fold into stable structures and provide essential biological functions are key goals in synthetic biology. In initial steps toward achieving these goals, we constructed a combinatorial library of de novo proteins designed to fold into 4-helix bundles. As described previously, screening this library for sequences that function in vivo to rescue conditionally lethal mutants of Escherichia coli (auxotrophs) yielded several de novo sequences, termed SynRescue proteins, which rescued four different E. coli auxotrophs. In an effort to understand the structural requirements necessary for auxotroph rescue, we investigated the biophysical properties of the SynRescue proteins, using both computational and experimental approaches. Results from circular dichroism, size-exclusion chromatography, and NMR demonstrate that the SynRescue proteins are α-helical and relatively stable. Surprisingly, however, they do not form well-ordered structures. Instead, they form dynamic structures that fluctuate between monomeric and dimeric states. These findings show that a well-ordered structure is not a prerequisite for life-sustaining functions, and suggests that dynamic structures may have been important in the early evolution of protein function.
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
authors.forename: Grant S
authors.initials: GS
authors.lastname: Murphy
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
authors.forename: Jack B
authors.initials: JB
authors.lastname: Greisman
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08540, USA. Electronic address: hecht@princeton.edu.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Hecht
mesh_terms: D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D050296:Microbial Viability; D008969:Molecular Sequence Data; D011487:Protein Conformation; D055503:Protein Multimerization; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011506:Proteins
keywords: artificial proteomes; de novo protein design; helix bundle; synthetic biology