De novo designed peptides for cellular delivery and subcellular localisation

Abstract

Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here, we describe the design, characterization, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterized in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes.

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles

“…37,39,[85][86][87] Moreover, because our designed peptides and protein assemble efficiently in cells, such as E. coli, we anticipate applications to intervene in and to augment natural sub-cellular processes. 10,53,58,62,88,89 In short, we believe that our work adds fundamental understanding of the structural principles and sequence-to-structure relationships for coiled coils generally and 4-helix bundles specifically; and that our new designs provide platforms for future de novo design, and chemical and synthetic biology programs.…”

“…37, 39, 85–87 Moreover, because our designed peptides and protein assemble efficiently in cells, such as E. coli , we anticipate applications to intervene in and to augment natural sub-cellular processes. 10, 53, 58, 62, 88, 89…”

“…41,[50][51][52][53][54][55] That all said, designing antiparallel CC assemblies from first principles has been more challenging. 33,[56][57][58] Moreover, subtle changes in primary sequence or even experimental conditions can induce switches from energetically close parallel assemblies to antiparallel conformations. 33,[59][60][61] For example, recently, we reported the rational redesign of an antiparallel CC tetramer, apCC-Tet, following the serendipitous discovery of up−down−up−down tetramers adopted by a point mutation of our original parallel hexamer, CC-Hex.…”

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles

“…37,39,85–87 Moreover, because our designed peptides and protein assemble efficiently in cells, such as E. coli , we anticipate applications to intervene in and to augment natural sub-cellular processes. 10,53,58,62,88,89…”

“…41,[50][51][52][53][54][55] That all said, designing antiparallel CC assemblies from rst principles has been more challenging. 33,[56][57][58] Moreover, subtle changes in primary sequence or even experimental conditions can induce switches from energetically close parallel assemblies to antiparallel conformations. 33,[59][60][61] For example, recently, we reported the rational redesign of an antiparallel CC tetramer, apCC-Tet, following the serendipitous discovery of up-downup-down tetramers adopted by point mutations in our original parallel hexamer, CC-Hex.…”