The E/K coil, a heterodimeric coiled-coil, has been designed as a universal peptide capture and delivery system for use in applications such as biosensors and as an expression and affinity purification tag. In this design, heterodimer formation is specified through the placement of charged residues at the e and g positions of the heptad repeat such that the E coil contains all glutamic acid residues at these positions, and the K coil contains all lysine residues at these positions. The affinity and stability of the E/K coil have been modified to allow a greater range of conditions for association and dissociation. Increasing the hydrophobicity of the coiled-coil core, by substituting isoleucine for valine, gave increases in stability of 2.81 and 3.73 kcal/mol (0.47 kcal/ mol/substitution). Increasing the ␣-helical propensity of residues outside the core, by substituting alanine for serine, yielded increases in stability of 2.68 and 3.28 kcal/mol (0.41 and 0.45 kcal/mol/substitution). These sequence changes yielded a series of heterodimeric coiledcoils whose stabilities varied from 6.8 to 11.2 kcal/mol, greatly expanding their scope for use in protein engineering and biomedical applications.The coiled-coil is an oligomerization domain found in a wide variety of proteins, including transcription factors, motor proteins, chaperone proteins, and viral fusion proteins (1-4). Recent surveys of genomic data bases suggest that up to 10% of eukaryotic proteins contain sequences predicted to be coiledcoils (5). This structural motif has been of considerable interest, both because of its diversity in structure and oligomerization state and because of its many advantages as a model system for protein design (6, 7). Coiled-coils contain a single type of secondary structure, the ␣-helix, which is easy to monitor experimentally by circular dichroism (CD) spectroscopy. Their quaternary interactions yield a structure that is folded stably in aqueous solution at neutral pH, unlike most singlestranded ␣-helices.The structural features of coiled-coils have been reviewed extensively (3,7,8). Their sequences are characterized by a heptad repeat, denoted abcdefg, in which positions a and d are occupied by hydrophobic residues. The side chains from the a and d residues pack against each other in a "knobs-intoholes" manner (9), forming a continuous hydrophobic core. Maintaining this packing along the length of the ␣-helices results in their wrapping around each other in a left-handed supercoil. The side chains of the residues in positions e and g lie alongside the hydrophobic core. These positions are typically occupied by charged residues that can participate in i to iЈϩ5 electrostatic interactions, which have been found to play an important role in specifying homo-and heteroassociation in native coiled-coils (1, 10 -13). The preference for electrostatic attractions over repulsions has been key to the de novo design of heterodimeric coiled-coils (14 -22).Despite the apparent simplicity of coiled-coils, their structures display a surpri...

The E/K coil, a heterodimeric coiled-coil, has been designed as a universal peptide capture and delivery system for use in applications such as biosensors and affinity chromatography. In this design, heterodimer formation is specified through the placement of charged residues at the e and g positions of the heptad repeat. The affinity and stability of the E/K coil has been modified in order to allow a greater range of conditions for association and dissociation by varying the chain length to obtain three, four and five heptad coiled-coils (21, 28 and 35 residues per polypeptide chain). The effect of chain length on stability and folding was examined by circular dichroism spectroscopy, guanidine hydrochloride denaturation, and redox equilibrium experiments. It was found that increases in chain length produced increases in the stability of heterodimeric coiled-coils, but in a nonlinear fashion. The resulting disulfide-bridged heterostranded molecules and reduced heterodimers span a wide range of stabilities (deltaG=3.3-11.9 kcal/mol), greatly expanding their scope for use in protein engineering and biomedical applications.

We have de novo designed a heterodimeric coiled-coil formed by two peptides as a capture/ delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coil (KVSALKE heptad sequence) and E coil (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 µM (E3/K3). From these K d values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the coiled-coils. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.The R-helical coiled-coil is an oligomerization domain that is naturally present in a wide variety of proteins such as transcription factors, cytoskeletal proteins, motor proteins, and viral fusion proteins (1-3). The structural properties of coiled-coils have been extensively characterized from numerous high-resolution crystal and NMR structures (3-5). The formation of coiled-coils requires the wrapping of two or more amphipathic R-helices around each other in a lefthanded supercoil fashion; the helices may be aligned in either a parallel or antiparallel manner. The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13-17). Their relatively small size, in addition to their well-defined structure, make coiled-coils a very attractive system for protein engineering and biotechnological applications (5, 18) such as the construction of miniaturized antibodies, the control of signaling protein domain oligomerization, and the specific targeting of GFP to cytoskeletal proteins (see ref 4 for ...