Coiled-coil proteins contain a characteristic seven-residue sequence repeat whose positions are designated a to g. The interacting surface between ␣-helices in a classical coiled coil is formed by interspersing nonpolar side chains at the a and d positions with hydrophilic residues at the flanking e and g positions. To explore how the chemical nature of these core amino acids dictates the overall coiled-coil architecture, we replaced all eight e and g residues in the GCN4 leucine zipper with nonpolar alanine side chains. Surprisingly, the alanine-containing mutant forms a stable ␣-helical heptamer in aqueous solution. The 1.25-Å resolution crystal structure of the heptamer reveals a parallel seven-stranded coiled coil enclosing a large tubular channel with an unusual heptad register shift between adjacent staggered helices. The overall geometry comprises two interleaved hydrophobic helical screws of interacting cross-sectional a and d layers that have not been seen before. Moreover, asparagines at the a positions play an essential role in heptamer formation by participating in a set of buried interhelix hydrogen bonds. These results demonstrate that heptad repeats containing four hydrophobic positions can direct assembly of complex, higher-order coiled-coil structures with rich diversity for close packing of ␣-helices.protein design ͉ protein structure ͉ helix-helix interfaces ͉ buried polar interactions ͉ cavity H elix-helix interactions are ubiquitous in the native structure of proteins and in associations among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signaling and transport. Coiled coils afford a unique model system for elucidating principles of molecular recognition between helices (1-4). The conformation of coiled coils is prescribed by a characteristic 7-aa repeat, the 3-4 heptad repeat denoted as a-b-c-d-e-f-g (5). Positions a and d are typically occupied by hydrophobic amino acids such as Leu, Ile, Val, and Ala, whereas residues at positions e and g are frequently polar or charged (5-8). Crick's (9) classical analysis proposed that the nonpolar a and d side chains associate by means of complementary ''knobs-into-holes'' packing to form supercoiled ␣-helical ribbons. Beyond the role of hydrophobic side chains at the core a and d positions, coiled coils can use intra-and interhelical electrostatic interactions to tune their stability, especially those between the flanking e and g positions of neighboring chains (10-18). Despite their seeming regularity in sequence and packing patterns, coiled coils exhibit remarkable diversity in the number and arrangement of the associating helices: two-to five-stranded structures have been identified with parallel or antiparallel helix orientation (2). The structural parameters of dimers have been described by Crick (9), and general rules governing the close packing of ␣-helices in hexamers and dodecamers have been deduced (19,20).Analysis of coiled coils provides insight into the general problem of protein folding ...

ABSTRACT:We report on the synthesis of poly(diethylene oxidealt-oxymethylene), P(2EO-MO), via cationic ring-opening polymerization of the cyclic ether monomer, 1,3,6-trioxocane. We use a combined experimental and computational approach to study ion transport in electrolytes comprising mixtures of P(2EO-MO) and lithium bis(trifluoromethanesulfonyl) imide (LiTFSI) salt. Mixtures of poly(ethylene oxide) (PEO) and LiTFSI are used as a baseline. The maximum ionic conductivities, σ, of P(2EO-MO) and PEO electrolytes at 90°C are 1.1 × 10 −3 and 1.5 × 10 −3 S/cm, respectively. This difference is attributed to the T g of P(2EO-MO)/ LiTFSI (−12°C), which is significantly higher than that of PEO/LiTFSI (−44°C) at the same salt concentration. Self-diffusion coefficients measured using pulsed-field gradient NMR (PFG-NMR) show that both Li + and TFSI − ions diffuse more rapidly in PEO than in P(2EO-MO). However, the NMR-based cation transference number in P(2EO-MO) (0.36) is approximately twice that in PEO (0.19). The transference number measured by the steady-state current technique, t +,ss , in P(2EO-MO) (0.20) is higher than in PEO (0.08) by a similar factor. We find that the product σt +,ss is greater in P(2-EO-MO) electrolytes; thus, P(2EO-MO) is expected to sustain higher steady-state currents under dc polarization, making it a more efficacious electrolyte for battery applications. Molecular-level insight into the factors that govern ion transport in our electrolytes was obtained using MD simulations. These simulations show that the solvation structures around Li + are similar in both polymers. The same is true for TFSI − . However, the density of Li + solvation sites in P(2EO-MO) is double that in PEO. We posit that this is responsible for the observed differences in the experimentally determined transport properties of P(2EO-MO) and PEO electrolytes.

We perform a joint experimental and computational study of ion transport properties in a systematic set of linear polyethers synthesized via acyclic diene metathesis (ADMET) polymerization. We measure ionic conductivity, σ, and glass transition temperature, T g, in mixtures of polymer and lithium bis­(trifluoro­methane­sulfonyl)­imide (LiTFSI) salt. While T g is known to be an important factor in the ionic conductivity of polymer electrolytes, recent work indicates that the number and proximity of lithium ion solvation sites in the polymer also play an important role, but this effect has yet to be systematically investigated. Here, adding aliphatic linkers to a poly­(ethylene oxide) (PEO) backbone lowers T g and dilutes the polar groups; both factors influence ionic conductivity. To isolate these effects, we introduce a two-step normalization scheme. In the first step, Vogel–Tammann–Fulcher (VTF) fits are used to calculate a temperature-dependent reduced conductivity, σr(T), which is defined as the conductivity of the electrolyte at a fixed value of T – T g. In the second step, we compute a nondimensional parameter f exp, defined as the ratio of the reduced molar conductivity of the electrolyte of interest to that of a reference polymer (PEO) at a fixed salt concentration. We find that f exp depends only on oxygen mole fraction, x 0, and is to a good approximation independent of temperature and salt concentration. Molecular dynamics simulations are performed on neat polymers to quantify the occurrences of motifs that are similar to those obtained in the vicinity of isolated lithium ions. We show that f exp is a linear function of the simulation-derived metric of connectivity between solvation sites. From the relationship between σr and f exp we derive a universal equation that can be used to predict the conductivity of ether-based polymer electrolytes at any salt concentration and temperature.

Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

Entry of SARS coronavirus into its target cell requires large-scale structural transitions in the viral spike (S) glycoprotein in order to induce fusion of the virus and cell membranes. Here we describe the identification and crystal structures of four distinct alpha-helical domains derived from the highly conserved heptad-repeat (HR) regions of the S2 fusion subunit. The four domains are an antiparallel four-stranded coiled coil, a parallel trimeric coiled coil, a four-helix bundle, and a six-helix bundle that is likely the final fusogenic form of the protein. When considered together, the structural and thermodynamic features of the four domains suggest a possible mechanism whereby the HR regions, initially sequestered in the native S glycoprotein spike, are released and refold sequentially to promote membrane fusion. Our results provide a structural framework for understanding the control of membrane fusion and should guide efforts to intervene in the SARS coronavirus entry process.