Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled coils

“Moreover, I am sure that this will grow with the application of AlphaFold2 to entire proteomes and the development of AlphaFold2 Multimer (3, 4, 242).”

“A particularly interesting example is the H protein from bacteriophage Φ X174, which forms a tube that is essential for the virus to infect E. coli and is believed to transfer the viral ssDNA across the host periplasm.”

“Moreover, I am sure that this will grow with the application of AlphaFold2 to entire proteomes and the development of AlphaFold2 Multimer (3, 4, 242).”

“As a consequence of steric constraints in polypeptide chains — as later formalized by Ramachandran in his famous plot (26, 27) — the α helix has precisely 3.6 residues per turn (Fig.”

“…long fi brous coiled coils in motor proteins and intermediate fi laments (IFs), which were among the fi rst targets in structural molecular biology (21, 224); and short oligomerization domains like the leucine zippers (5), which became a model for protein folding, structure, and design (6, 7, 73).”

“Moreover, I am sure that this will grow with the application of AlphaFold2 to entire proteomes and the development of AlphaFold2 Multimer (3, 4, 242).”

“The hemagglutinin story became more interesting as it unraveled; for example, revealing a spring-loaded switch to a longer trimeric CC leading to virus-host membrane fusion, Figure 1 D (46, 47), and targets for drug design (48, 49).”

“The eukaryotic SNARE and related proteins involved in intracellular vesicular traf fi cking also promote membrane fusion, though through a very different mechanism (267 – 270).”

“However, these cores are very different from those of globular proteins (6, 19, 37, 45, 50).”

“The coiled coil came out of physics: Francis Crick ’ s description of the coiled coil, as he fi rst named it, is mathematical (22, 23).”

“The hemagglutinin story became more interesting as it unraveled; for example, revealing a spring-loaded switch to a longer trimeric CC leading to virus-host membrane fusion, Figure 1 D (46, 47), and targets for drug design (48, 49).”

“…long fi brous coiled coils in motor proteins and intermediate fi laments (IFs), which were among the fi rst targets in structural molecular biology (21, 224); and short oligomerization domains like the leucine zippers (5), which became a model for protein folding, structure, and design (6, 7, 73).”

“I have not touched on this in my perspective, but there are excellent reviews from these groups (110, 111, 285 – 287).”

“The reason for this is apparent in the X-ray crystal structure of GCN4-p1 dimer, wherethe Asnpaircanmakeaside-chainhydrogenbond, Figure 3 F (6).”

“The octameric barrel of Wza is interesting as this is a different solution to spanning the outer bacterial membrane than seeing the abundant β -porin – based domains, as seen in TolC, Figure 5 D (103).”

“The hemagglutinin story became more interesting as it unraveled; for example, revealing a spring-loaded switch to a longer trimeric CC leading to virus-host membrane fusion, Figure 1 D (46, 47), and targets for drug design (48, 49).”

“…long fi brous coiled coils in motor proteins and intermediate fi laments (IFs), which were among the fi rst targets in structural molecular biology (21, 224); and short oligomerization domains like the leucine zippers (5), which became a model for protein folding, structure, and design (6, 7, 73).”

“…for predicting, building, analyzing, and visualizing coiled-coil structures Prediction Several tools for predicting coiled-coil structure from sequence are brought together and implemented at Andrei Lupas ’ s Max Planck Institute Bioinformatics Toolkit (59) (https://toolkit.tuebingen.mpg.de/).”

“They result in further left-handed, some right-handed, and even “ straight ” coiled coils (19, 55 – 58).”

“The hemagglutinin story became more interesting as it unraveled; for example, revealing a spring-loaded switch to a longer trimeric CC leading to virus-host membrane fusion, Figure 1 D (46, 47), and targets for drug design (48, 49).”

“Moreover, they have been used widely as rules for protein design by many groups to deliver many de novo – designed coiled-coil peptides and proteins (12, 14, 51, 70, 80), which are described in more detail below.”

“The old view that coiled coils are simply oligomerization domains and spacers changed with improved abilities to spot coiled-coil sequences in proteins using computational biology (227), see Box 2.”

“This followed the pioneering work of Regan and DeGrado and by Hecht and the Richardsons (149, 150).”

“In coiled coils, the KIH packing is tight and intimate; whereas, in globular proteins, the side chains pack more loosely (71).”

“As reviewed elsewhere (70, 146, 147), these have been design targets for DeGrado, Dutton, their former group Left-hand column, systematic name of the de novo coiled-coil assembly (Fig.”

“…variants of these, hSAFs (165); the templated assembly of bacterial collagens (166); the design of Self-Assembled peptide caGEs, SAGEs (167) and the applications of these in cell delivery and vaccine development (168, 169); the construction of arti fi cial transcription factors (119,…”

“These include the following: Lupas ’ s original COILS program (60) (now PCOIL), DeepCoil (61), and MARCOIL (62).”

“…the following: the construction and application of Self-Assembled peptide Fi-bers, SAFs (164), and hydrogel-forming variants of these, hSAFs (165); the templated assembly of bacterial collagens (166); the design of Self-Assembled peptide caGEs, SAGEs (167) and the applications of these in…”

“Examination of the high-resolution structures of the two series of peptide assemblies — namely, Harbury ’ s engineered GCN4-p1 peptides and a set of de novo design peptides (73, 74, 89) — reveals that something more is going on.”

“…the apparent simplicity of the leucine zippers belies an underlying complexity, which includes many proteins in humans, the speci fi cation of homo-and hetero-oligomers between these, and differing levels of expression, which combine to give exquisite control over transcription (137, 142, 225, 226).”

“In 1991, O ’ Shea, Alber, and Kim determined the fi rst atomic-resolution coiled-coil structure for the leucine-zipper peptide of the yeast transcriptional activator GCN4 (6), Figure 1 A .”

“…been proposed, structural studies of an archaeal HAMP suggest that a ‘ gearbox ’ mechanism could be a play in which alternate coiled-coil core-packing arrangements drive the helices to rotate about their long axes to generate a rotational signal that is passed to the intracellular components (273).”

“And the transcriptional effector TAZ undergoes LLPS via its coiled-coil domain in the nucleus of human breast cancer cells to recruit transcription machinery and promote gene expression (261).”

“I have already mentioned the α HB structures of E. coli Wza (244) and the F 1 F 0 ATP synthase (104), Figure 5, E and G .”

“Nevertheless, since their discovery, Harbury ’ s basic sequence-to-structure relationships have been con fi rmed by analyses of many natural coiled-coil sequences and structures (37, 50, 75) and through quantitative biophysical studies (78, 79).”

“…apCC-Di-AB, CC-Tri, CC-Tet, and related peptides include the following: the construction and application of Self-Assembled peptide Fi-bers, SAFs (164), and hydrogel-forming variants of these, hSAFs (165); the templated assembly of bacterial collagens (166); the design of Self-Assembled peptide…”

“Later, low-resolution 15 Å (43) and 7 Å (44) structures from EM and X-ray diffraction revealed a supercoiled pair of α helices, Figure 1 B .”

“Speci fi cally on the latter, the DeGrado and Baker groups have used sequence-to-structure relationships garnered from the α HBs to stabilize pentameric bundles of phospholamban (218) and in the de novo design of ion channels (219), respectively.”

“The coiled coil came out of physics: Francis Crick ’ s description of the coiled coil, as he fi rst named it, is mathematical (22, 23).”

“Nonetheless, these systems are being put to good use in a variety of applications, including the following: driving membrane fusion (127, 128); directing the patterned aggregation of bacterial and human cells (129); developing peptide origami by Jerala (65, 80, 130); (89).”

“The human PDZ-and coiled coil – containing protein harmonin from the sensory cells of the inner ear and retina undergoes LLPS at high concentrations in vitro .”

“As reviewed elsewhere (70, 146, 147), these have been design targets for DeGrado, Dutton, their former group Left-hand column, systematic name of the de novo coiled-coil assembly (Fig.”

“…the apparent simplicity of the leucine zippers belies an underlying complexity, which includes many proteins in humans, the speci fi cation of homo-and hetero-oligomers between these, and differing levels of expression, which combine to give exquisite control over transcription (137, 142, 225, 226).”

“Some allow coiled-coil backbones to be built quickly and accurately (28, 31), and others allow full atomistic modeling of coiled coils including side chains (29, 33, 35, 36).”

“Nonetheless, these systems are being put to good use in a variety of applications, including the following: driving membrane fusion (127, 128); directing the patterned aggregation of bacterial and human cells (129); developing peptide origami by Jerala (65, 80, 130); (89).”

“Thus, the coiled coil is inherently parametric, which makes it physics, predictable, modelable and, as, we will see, ultimately designable (28).”

“I have not touched on this in my perspective, but there are excellent reviews from these groups (110, 111, 285 – 287).”

“While de novo protein design has been active for ≈ 40 years, the fi eld is now advancing rapidly and booming (51, 70, 110 – 112).”

“From that, rational and computational peptide/protein design and engineering has delivered many examples of these, Figures 4 and 5, A and B (33, 34, 40, 100, 138, 144).”

“In turn, this would allow the modules to be used reliably in synthetic biology to construct more complex and functional protein-like objects (114, 115).”

“Exploring other sequence repeats and mixing and matching those available to increase sequence complexity may help address this issue; indeed, others like Mason, Keating, and Jerala are showing the way here (93, 124, 163).”

“A prime example of this is the conservation of an Asn residue at a central a site in the wider family of leucine-zipper transcription factors (86).”

“Nevertheless, since their discovery, Harbury ’ s basic sequence-to-structure relationships have been con fi rmed by analyses of many natural coiled-coil sequences and structures (37, 50, 75) and through quantitative biophysical studies (78, 79).”

“I have already mentioned the α HB structures of E. coli Wza (244) and the F 1 F 0 ATP synthase (104), Figure 5, E and G .”

“However, Pauling et al. considered other repeats in α -helical conformations, speci fi cally, those with 11, 15, and 18 residues (25, 54).”

“Coiled-coil function has been surveyed in good reviews over the years (8 – 11 , 13 )”

“…systems, including the following: the aforementioned designs from O ’ Shea and Kim, “ peptide Velcro ” (116), and from Litowski and Hodges, “ E/K coil ” peptides (117, 118); Keating ’ s “ SYN-ZIP ” designs (123, 124); and the sets of coiled-coil hetero-dimers from Jerala (91, 125) and Mason (93).”

“…important biophysical properties such as coiled-coil dynamics, exchange, and turnover; moving beyond coiled coils as simple oligomerization domains and spacers and exploring the complexity of coiled-coil architectures in the context of larger and functional biological assemblies (235, 240).”

“Indeed, we have observed this for CC-Tet and some α HB systems (89, 100, 109, 144, 145, 181), and others have reported changes in oligomer states when incorporating some of the modules into larger fusion proteins (197, 198).”

“These include the following: Lupas ’ s original COILS program (60) (now PCOIL), DeepCoil (61), and MARCOIL (62).”

“However, as has been known for some time (246, 247), there is a lot more to these coiled-coil proteins than the simple text-book picture portrays.”

“As a consequence of steric constraints in polypeptide chains — as later formalized by Ramachandran in his famous plot (26, 27) — the α helix has precisely 3.6 residues per turn (Fig.”

“Exploring other sequence repeats and mixing and matching those available to increase sequence complexity may help address this issue; indeed, others like Mason, Keating, and Jerala are showing the way here (93, 124, 163).”

“This principle has been exploited to drive membrane fusion in a variety of synthetic and biological membrane systems by Kros et al. (127, 128).”

“The history and achievements of this sub fi eld are well documented (12, 14, 51, 70, 80), so I will not repeat it here.”

“Possibilities here include nature ’ s inclusion of nonheptad sequence repeats (55, 56) and so-called trigger sequences for folding (277 – 279).”

“However, given the hyperthermal stability possible with even relatively short coiled coils (33, 34, 85), the disruption of perfect hydrophobic repeats is almost certainly essential for protein dynamics and turnover in natural coiled coils.”

“In 1972, Sodek et al. reported the partial sequence of rabbit tropomyosin (42), which was anticipated to be an extended dimeric coiled coil.”

“Smaller transmembrane barrels include the ion channel phospholamban, which is a pentamer as shown by a combined solution and solid-state NMR structure determined in lipid bilayers, Figure 5 H (272).”

“While on altered and dynamic packing arrangements in natural coiled coils, I should mention Ghosh ’ s work on the surface-associated M proteins of Streptococcus pyogenes (Strep A) (274).”

“…peptide synthesis or as fusions to proteins of interest via gene synthesis and recombinant expression; (ii) they can be adapted to render a range of association constants from μ M to pM (92, 183); and (iii) in several cases, multiple orthogonal pairs have been engineered (91 – 94, 124, 182).”

“Speci fi cally on the latter, the DeGrado and Baker groups have used sequence-to-structure relationships garnered from the α HBs to stabilize pentameric bundles of phospholamban (218) and in the de novo design of ion channels (219), respectively.”

“As discussed in previous sections, α HBs have expanded our understanding and our reach into coiled-coil structural space, and they have inspired new protein designs (13, 140, 245).”

“Nevertheless, since their discovery, Harbury ’ s basic sequence-to-structure relationships have been con fi rmed by analyses of many natural coiled-coil sequences and structures (37, 50, 75) and through quantitative biophysical studies (78, 79).”

“Our adaptations of the α HBs have included the following: assembling peptide-based fi bers and nanotubes (176, 177), following the approach of Conticello (178); installing a Cys-His-Glu catalytic triad in the channel of CC-Hept to effect ester hydrolysis; developing receptors for reporters dyes and…”

“…de novo coiled-coil modules have been CC-Di-AB (92) and related heterodimeric systems, such as Hodges ’ E/K peptides (117, 118), Keating ’ s SYNZIP systems (124), and others from Jerala (91, 94), Mason (93, 182), O ’ Shea JBC REVIEWS: Understanding α -helical coiled coils (116), and Vinson (183).”

“This principle has been exploited to drive membrane fusion in a variety of synthetic and biological membrane systems by Kros et al. (127, 128).”

“PDB, Protein Data Bank. as “ peptide-PAINT ” or “ live-PAINT ” for high-resolution light microscopy (131, 132); and targeting natural coiled coils in vitro and in cells (133 – 137).”

“These coupled with genome sequencing have led to coiled-coil predictions for whole genomes (228 – 233).”

“Moreover, they have been used widely as rules for protein design by many groups to deliver many de novo – designed coiled-coil peptides and proteins (12, 14, 51, 70, 80), which are described in more detail below.”

“…aromatic cores that might be suitable for electron transfer (82, 83); to direct the assembly of reversible protein condensates in living cells (213, 214); to encapsulate carbon nanotubes with these water-soluble peptides (215); to explore valency effects in natural protein – protein interactions,…”

“…of these in cell delivery and vaccine development (168, 169); the construction of arti fi cial transcription factors (119, 170) and cytoscaffolds (171) that assemble and function in bacterial cells; the directed assembly of large DNA origamis (172); the presentation of functioning hotspot…”

“Crick ’ s formalism has been con fi rmed by numerous experiments and analyses over the past 4 to 5 decades (20, 41), as illustrated by the following.”

“Previously, with Alber, we had used the idea of electrostatic hetero-speci fi cation in computational design to make a hetero-trimer, CC-Tri-ABC (120), a structure for which was later determined by X-ray crystallography, Figure 4 (121).”

“…to explore valency effects in natural protein – protein interactions, e.g. , galectin-glycan binding (216); to develop Spy&Go, augmenting the widely used SpyCatcher system through multi-merization (217); and to facilitate the engineering and de novo design of transmembrane proteins (218, 219).”

“Exploring other sequence repeats and mixing and matching those available to increase sequence complexity may help address this issue; indeed, others like Mason, Keating, and Jerala are showing the way here (93, 124, 163).”

“Speci fi cally on the latter, the DeGrado and Baker groups have used sequence-to-structure relationships garnered from the α HBs to stabilize pentameric bundles of phospholamban (218) and in the de novo design of ion channels (219), respectively.”

“Coiled-coil function has been surveyed in good reviews over the years (8 – 11 , 13 )”

“Such an article would be redundant because many excellent reviews are already available (8 – 14), as are others covering newer topics such as the applications of natural and designed coiled coils in biotechnology and synthetic biology (15 – 18).”

“With some tweaks, the main tenets of Crick ’ s model are evident and validated by these structures and analyses (19, 20, 37, 50).”

“For introductions to biomolecular condensation and phase separation in nature and synthetic biology, two excellent reviews have been published in a special issue of Nature Chemical Biology (254, 255).”

“Nonetheless, these systems are being put to good use in a variety of applications, including the following: driving membrane fusion (127, 128); directing the patterned aggregation of bacterial and human cells (129); developing peptide origami by Jerala (65, 80, 130); (89).”

“Wza has inspired the design of a peptide-based ion channel (271).”

“…of the α HBs have included the following: assembling peptide-based fi bers and nanotubes (176, 177), following the approach of Conticello (178); installing a Cys-His-Glu catalytic triad in the channel of CC-Hept to effect ester hydrolysis; developing receptors for reporters dyes and…”

“Possibilities here include nature ’ s inclusion of nonheptad sequence repeats (55, 56) and so-called trigger sequences for folding (277 – 279).”

“…called these Type III interfaces, which are manifested in large helical barrels such as the 12-helix assembly of TolC (103) and more recent structures of the F 1 F 0 ATP synthase (104); and they are being used in design by Conticello et al. to design fi brous and nanotubular assemblies (105 – 107).”

“Indeed, we have observed this for CC-Tet and some α HB systems (89, 100, 109, 144, 145, 181), and others have reported changes in oligomer states when incorporating some of the modules into larger fusion proteins (197, 198).”

“The eukaryotic SNARE and related proteins involved in intracellular vesicular traf fi cking also promote membrane fusion, though through a very different mechanism (267 – 270).”

“F , the H protein from the Φ X174 coliphage forms a 10-stranded α - helical tube, which can span the periplasm of the host to deliver its ssDNA genome (4jpp (291)).”

“All images were made in PyMol (pymol.org) using the PDB codes given or from models generated in CCBuilder/ISAMBARD (29, 35, 36).”

“…the apparent simplicity of the leucine zippers belies an underlying complexity, which includes many proteins in humans, the speci fi cation of homo-and hetero-oligomers between these, and differing levels of expression, which combine to give exquisite control over transcription (137, 142, 225, 226).”

“Nevertheless, since their discovery, Harbury ’ s basic sequence-to-structure relationships have been con fi rmed by analyses of many natural coiled-coil sequences and structures (37, 50, 75) and through quantitative biophysical studies (78, 79).”

“…antiparallel coiled-coil tetramers: the use of a = d = Leu or better a = Leu d = Ile cores; an obligate Ala at e , similar to so-called Alacoils (151); a preference for Gln at g ; and the use of charge complementarity at b & c as a fi nal guide to helix-helix speci fi cation and orientation, and…”

“In turn, this would allow the modules to be used reliably in synthetic biology to construct more complex and functional protein-like objects (114, 115).”

“The idea that residues at e and g sites progressively become involved in coiled-coil interfaces with increasing oligomeric state was fi rst formalized by Wal-shaw (96, 97), though Dunker and Zaleske considered the more general problem earlier (98), as did DeGrado et al. (99) at about the same time as Walshaw.”

“However, the following are currently available: LOGICOIL (63) (http://coiledcoils.chm.bris.ac.uk/LOGICOIL/) and Multicoil2 (64) (http://cb.csail.mit.edu/cb/multicoil2/cgi-bin/ multicoil2.cgi).”

“Despite the apparent rigidity and stability of these IF coiled-coil assemblies, regions of the structure have been shown to be more labile, which may impact on folding, assembly, function, and misfunction (252, 253).”

“Nevertheless, since their discovery, Harbury ’ s basic sequence-to-structure relationships have been con fi rmed by analyses of many natural coiled-coil sequences and structures (37, 50, 75) and through quantitative biophysical studies (78, 79).”

“…too (see below); to construct arti fi cial cages by appending CC-Tri, CC-Tet, or CC-Pent to natural proteins with complementary symmetries (199 – 202); to form de fi ned oligomers of photosynthetic reaction centers in membranes (203); to display multiple sugar moieties (204), small-molecule –…”

“Estimates across all genomes suggest ≈ 0.5 to 6.5% of protein sequences contain coiled-coil regions (232).”

“…factors in Escherichia coli (193); designing molecular switches (beacons) for detecting natural proteins (194); masking therapeutic antibodies that can be activated by proteolysis of the coiled coils (195); and appending them to natural bacterial proteins to direct fi ber formation (196).”

“However, the following are currently available: LOGICOIL (63) (http://coiledcoils.chm.bris.ac.uk/LOGICOIL/) and Multicoil2 (64) (http://cb.csail.mit.edu/cb/multicoil2/cgi-bin/ multicoil2.cgi).”

“Thus, the hexamer and heptamer open up potential “ dark-matter ” protein structures to explore and exploit (139, 140).”

“Many others have developed heterodimeric AB systems, including the following: the aforementioned designs from O ’ Shea and Kim, “ peptide Velcro ” (116), and from Litowski and Hodges, “ E/K coil ” peptides (117, 118); Keating ’ s “ SYN-ZIP ” designs (123, 124); and the sets of coiled-coil hetero-dimers from Jerala (91…”

“This plurality of heterodimeric modules has helped advance synthetic biology, vesicle/proto-cell work, and protein origami, as reviewed elsewhere (15, 16).”

“As a result, true and robust sequence-to-structure relationships are dif fi cult to decipher (37, 161).”

“However, as has been known for some time (246, 247), there is a lot more to these coiled-coil proteins than the simple text-book picture portrays.”

“Indeed, the ≈ 30-residue leucine-zipper domains are half the size of even the smallest recognized globular proteins and on a par with so-called miniproteins, which are a niche type of protein (72).”

“Moving out of the nucleus, the coiled-coil golgi protein GM130 and other members of the golgin family have been shown to undergo LLPS in vitro and when overexpressed in HeLa cells (262, 265).”

“Indeed, although aromatic residues can be introduced into both natural and de novo coiled-coil peptides, they tend to result in unusual structures that go beyond the classical and symmetric dimers, trimers, and tetramers (81 – 84).”

“Such an article would be redundant because many excellent reviews are already available (8 – 14), as are others covering newer topics such as the applications of natural and designed coiled coils in biotechnology and synthetic biology (15 – 18).”

“…and functional proteins (206, 207) on oligo-meric protein scaffolds; in combination with metal coordination, to drive supramolecular assembly (208, 209); to design metalloproteins (210); and to explore the fundamentals of coiled-coil assembly (211), including the incorporation of nonproteinogenic…”

“Our adaptations of the α HBs have included the following: assembling peptide-based fi bers and nanotubes (176, 177), following the approach of Conticello (178); installing a Cys-His-Glu catalytic triad in the channel of CC-Hept to effect ester hydrolysis; developing receptors for reporters dyes and…”

“…coils, progress is being made here by grafting hotspot residues from natural protein – protein interactions onto nat-ural and de novo – designed coiled-coil peptides (173, 282) and by using de novo – designed, single-chain coiled coils ( “ Alpha-bodies ” ) to present binding-site libraries (283).”

“…at e , similar to so-called Alacoils (151); a preference for Gln at g ; and the use of charge complementarity at b & c as a fi nal guide to helix-helix speci fi cation and orientation, and speci fi cally, using oppositely charged residues in the N-and C-terminal halves of these designs (152 – 154).”

“…displacement in peptide – peptide interactions (189); exploring the mechanics of biomaterials using biomolecular simulations and experiments (190 – 192); engineering hybrid (natural plus designed) arti fi cial transcription factors in Escherichia coli (193); designing molecular switches (beacons)…”

“…to effect ester hydrolysis; developing receptors for reporters dyes and lipophilic biomarkers that bind within the channels with μ M af fi nities (179) and using this as the basis for a differential biosensor (180); redesigning water-soluble α HBs to produce a membrane-spanning ion channel with…”

“PDB, Protein Data Bank. as “ peptide-PAINT ” or “ live-PAINT ” for high-resolution light microscopy (131, 132); and targeting natural coiled coils in vitro and in cells (133 – 137).”

“…polyproline-II helix followed packing with an α helix (290); three hetero-oligomeric coiled coils formed from acidic (A, red ) and basic (B, blue ) peptide chains (92, 119); an 8-helix bundle formed exclusively from 3 10 helices (52); and a single-chain 4-helix coiled coil based on apCC-Tet* (148).”

“Applications by others of CC-Di-AB include the following: designing self-sorting heterodimers (184); developing a fl uorescence-quencher pair that reports on coiled-coil stability and orientation (185) and, similarly, coassembled fl uorescent proteins (186); constructing synthetic virus-like capsids…”

“Of course, along with other challenges (280, 281), rigid coiled-coil scaffolds may not be optimal for binding.”

“Possibilities here include nature ’ s inclusion of nonheptad sequence repeats (55, 56) and so-called trigger sequences for folding (277 – 279).”

“I would like to fi nish with an example of a coiled coil as a molecular ruler (276).”

“Some allow coiled-coil backbones to be built quickly and accurately (28, 31), and others allow full atomistic modeling of coiled coils including side chains (29, 33, 35, 36).”

“PDB, Protein Data Bank. as “ peptide-PAINT ” or “ live-PAINT ” for high-resolution light microscopy (131, 132); and targeting natural coiled coils in vitro and in cells (133 – 137).”

“…at e , similar to so-called Alacoils (151); a preference for Gln at g ; and the use of charge complementarity at b & c as a fi nal guide to helix-helix speci fi cation and orientation, and speci fi cally, using oppositely charged residues in the N-and C-terminal halves of these designs (152 – 154).”

“…transcription factors (119, 170) and cytoscaffolds (171) that assemble and function in bacterial cells; the directed assembly of large DNA origamis (172); the presentation of functioning hotspot residues from natural protein – protein interactions (173); the direction of protein – protein…”

“Previously, with Alber, we had used the idea of electrostatic hetero-speci fi cation in computational design to make a hetero-trimer, CC-Tri-ABC (120), a structure for which was later determined by X-ray crystallography, Figure 4 (121).”

“Some allow coiled-coil backbones to be built quickly and accurately (28, 31), and others allow full atomistic modeling of coiled coils including side chains (29, 33, 35, 36).”

“Finally, in an impressive series of studies, Champion et al. have shown that the original CC-Hex sequence can be complexed with IgG to promote the cellular uptake and cytosolic delivery of these antibodies (220 – 223).”

“This was motivated by much of the work to that point being done on the GCN4-p1 system, which increasingly revealed contexts and alternate states that thwarted systematic studies (73, 75, 113).”

“…from ≈ 5 to 10 Å. Bottom row from left to right : a monomeric single-chain miniprotein with a polyproline-II helix followed packing with an α helix (290); three hetero-oligomeric coiled coils formed from acidic (A, red ) and basic (B, blue ) peptide chains (92, 119); an 8-helix bundle formed…”

“The resulting helices are no longer simple amphiphiles, they are bifaceted with the potential to form high-order structures (97, 99, 101, 102).”

“The idea that residues at e and g sites progressively become involved in coiled-coil interfaces with increasing oligomeric state was fi rst formalized by Wal-shaw (96, 97), though Dunker and Zaleske considered the more general problem earlier (98), as did DeGrado et al. (99) at about the same time as Walshaw.”

“…coils, progress is being made here by grafting hotspot residues from natural protein – protein interactions onto nat-ural and de novo – designed coiled-coil peptides (173, 282) and by using de novo – designed, single-chain coiled coils ( “ Alpha-bodies ” ) to present binding-site libraries (283).”

“Panels B and C are adapted from Walshaw and Woolfson (2003) (96) with permission. structures parametrically and energetically (32, 37 – 39).”

“…residues at e and g sites progressively become involved in coiled-coil interfaces with increasing oligomeric state was fi rst formalized by Wal-shaw (96, 97), though Dunker and Zaleske considered the more general problem earlier (98), as did DeGrado et al. (99) at about the same time as Walshaw.”

“…direct the assembly of reversible protein condensates in living cells (213, 214); to encapsulate carbon nanotubes with these water-soluble peptides (215); to explore valency effects in natural protein – protein interactions, e.g. , galectin-glycan binding (216); to develop Spy&Go, augmenting the…”

“Generalizing this idea, a systematic exploration of alpha-helical coiled-coils led to the design of novel architectures and development of principles to exploit these architectures for diverse functions 55 .”

“There is a rich history of designing coiled coils using simple geometric principles; this extensibility and designability have made them widely used constituents of designed protein materials 15 .”

“Many efforts have been focused on peptides folding into β-sheets (amyloid-like structures [22]) and also on the rational design of α-helical folding (proteins/peptide stabilization) [23].”