Two stages in the rational redesign of a peptide‐based, self‐assembling fiber (SAF) are described. The SAF system comprises two peptides designed to form an offset α‐helical coiled‐coil heterodimer. The “sticky‐ends” are complementary and promote longitudinal assembly. Alone, the two peptides are unstructured, but co‐assemble upon mixing to form α‐helical fibrils, which bundle to form fibers 40–50 nm wide and tens of micrometers long. Assembly is controllable and occurs at pH 7 in water, making SAFs a potential scaffold for 3D cell culture. The purposes of the redesigns were 1) to investigate the fiber‐thickening process, and 2) to increase fiber stability for potential biological and biomedical applications. First, mutations were made to the original peptide designs to increase fibril–fibril interactions and so produce thicker and more‐stable fibers. The second iteration aimed to increase the primary peptide–peptide interactions by increasing the overlap in the offset dimer and so promote the initial step in fiber formation. As judged by circular dichroism spectroscopy and transmission electron microscopy, both iterations improved fiber assembly and stability: the critical peptide concentration for assembly improved from 60 μM to 4 μM; the midpoint of thermal unfolding increased from 22 °C to 65 °C; and the salt tolerance improved from 75 mM to greater than 250 mM KCl. These improvements bring closer applications of the SAF system under physiological conditions, for example as a biocompatible material for 3D cell culture. In addition, ordered surface features were observed in the second‐ and third‐generation fibers compared with the original design. This indicates improved internal order in the redesigned fibers. In turn, this suggests a molecular mechanism for the improved stability and sheds light on the fiber‐assembly process.

We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of α-helices and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.

The construction of useful functional biomolecular components not currently part of the natural repertoire is central to synthetic biology. A new light-capturing ultra-high-efficiency energy transfer protein scaffold has been constructed by coupling the chromophore centers of two normally unrelated proteins: the autofluorescent protein enhanced green fluorescent protein (EGFP) and the heme-binding electron transfer protein cytochrome b(562) (cyt b(562)). Using a combinatorial domain insertion strategy, a variant was isolated in which resonance energy transfer from the donor EGFP to the acceptor cyt b(562) was close to 100% as evident by virtually full fluorescence quenching on heme binding. The fluorescence signal of the variant was also sensitive to the reactive oxygen species H(2)O(2), with high signal gain observed due to the release of heme. The structure of oxidized holoprotein, determined to 2.75 Å resolution, revealed that the two domains were arranged side-by-side in a V-shape conformation, generating an interchromophore distance of ~17 Å (14 Å edge-to-edge). Critical to domain arrangement is the formation of a molecular pivot point between the two domains as a result of different linker sequence lengths at each domain junction and formation of a predominantly polar interdomain interaction surface. The retrospective structural analysis has provided an explanation for the basis of the observed highly efficient energy transfer through chromophore arrangement in the directly evolved protein scaffold and provides an insight into the molecular principles by which to design new proteins with coupled functions.

Superficially similar cleavable targeting signals specify whether lumenal proteins are transported across the thylakoid membrane by a Sec-or v vpH-dependent pathway. A twin-arginine motif is essential but not sufficient to direct v vpHdependent targeting; here we show that a second determinant is located in the hydrophobic region. A highly hydrophobic amino acid is found either two or three residues C-terminal to the twinarginine in all known transfer peptides for the v vpH-dependent system, and substitution of this residue in the 23-kDa (23K) peptide markedly inhibits translocation. Further, whereas the insertion of twin-arginine in a Sec-dependent precursor does not permit efficient v vpH-dependent targeting, the simultaneous presence of a leucine at the +3 position (relative to the RR) enables the peptide to function as efficiently as an authentic transfer peptide. RRNVL, RRAAL and RRALA within a Sec targeting signal all support efficient v vpH-dependent targeting, RRNVA is less effective and RRNAA/RRNAG are totally ineffective. We conclude that the core signal for this pathway is a twin-arginine together with an adjacent hydrophobic determinant.z 1998 Federation of European Biochemical Societies.