A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology

Abstract

Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

“…This is consistent with literature reports of GCN4-based sequences, which have a = Val plus d = Leu and show a wide range of oligomer states, 13,[48][49][50] and with other design studies. 8,51,52 By contrast, CC-pII-I17N was markedly less folded and less thermally stable than any of the other peptides from either series, Table 1: it was only ≈50% helical at 10 µM concentration and 5˚C; and the TM was 36˚C, Figure S5B. Nonetheless, AUC data were still most compatible with a trimeric assembly, Figure S4J and Table 1; albeit with lowered apparent oligomer state, presumably because of the reduced TM.…”

“…2,16,18,19 This is also exploited in coiled-coil design: judicious placement of complementary charges-e.g., Lys-Glu pairs at e and g-can be used to pattern sequences to control homotypic or heterotypic assembly of peptides otherwise possessing the same core a & d residues. [19][20][21][22] Together, such heuristics have been used to design a suite of coiled-coil components, 8,[23][24][25] which have been used as modular building blocks in various synthetic-biology applications. [26][27][28][29][30] The above said, the a/d interfaces are not the exclusive province of hydrophobic residues; indeed, ≈1/4 of residues at these interfaces of structurally defined coiled coils are polar.…”

Installing hydrolytic activity into a completely de novo protein framework

“…Because of concerns over helix fraying, we discounted mutations near the termini and focused on those in the third heptad repeat. Using CCBuilder 38 and structural parameters from the crystal structures of a range of de novo coiled coils, 35,39 we generated in silico all-atom models for single, double and triple mutants as tetramers to heptamers (SI Table 1.1-1). Encouragingly, for 3 of the 5 sequences heptamer was predicted as the most-stable state.…”

“…Therefore, we used the homodimer, CC-Di, 39 incorporating Cys, His and Glu at sequential f, c and f positions ( Table 1). This peptide was highly -helical, soluble and dimeric ( SI Fig 1.3-9&10; SI Fig 1.4-5).…”

Structural and Functional Modeling of Artificial Bioactive Proteins

“…Recent progress in protein biochemistry and biophysics has enabled the construction of artificial (de novo) proteins with specific properties [1][2][3]. The predominant part of the possible protein sequences and structures not tested by evolution may be evaluated by de novo protein design, which could provide solutions to new protein-structure/function targets [2][3][4][5][6]. The goal of designing de novo proteins is to construct the molecules that structurally and functionally mimic natural proteins and to discover new structure-function relationships compared with those found in nature [2,6].…”