The preparation of synthetic molecules showing the remarkable efficiencies characteristic of natural biopolymer catalysts remains a formidable challenge for chemical biology. Although significant advances have been made in the understanding of protein structure and function, the de novo construction of such systems remains elusive. Re-engineered natural enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups, have been successful in catalysing a number of chemical transformations, sometimes with impressive efficiencies. But efforts to produce wholly synthetic catalytic peptides have typically resulted in compounds with questionable structural stability, let alone reactivity. Here we describe a 33-residue synthetic peptide, based on the coiled-coil structural motif, which efficiently catalyses the condensation of two shorter peptide fragments with high sequence- and diastereoselectivity. Depending on the substrates used, we observe rate enhancements of tenfold to 4,100-fold over the background, with catalytic efficiencies in excess of 10(4). These results augur well for the rational design of functional peptides.

Designed coiled-coil heterotrimers are described whose assembly is governed by both hydrophobic and hydrophilic forces. Sterically matched hydrophobic core side-chain packing of alanine and cyclohexylalanine has been shown to promote formation of a 1:1:1 heterotrimer. Manipulation of hydrophilic glutamic acid (Glu)/lysine (Lys) pairs at each of three helical contact interfaces provides a secondary recognition mechanism. Peptides with matched cores and hydrophilic contacts form stable heterotrimers (DeltaG(unf) at 25 degrees C = 17.93 kcal/mol; MW(app) = 11362 vs 11563 calcd for trimer), as do those with a single Lys/Lys (but not Glu/Glu) interface. The additional specificity engendered by simultaneous operation of two interfaces was used to design a system in which six different peptides are mixed to form three specific and independent heterotrimers in the same solution.

We describe the design and exploration of new buried polar groups to control coiled-coil dimerization. Employing our recently described method for on-resin guanidinylation, we have prepared coiled-coil peptides with a single core guanidine, spaced from the backbone by 1-3 methylene groups. Heterodimeric mixtures of these sequences with guanidine, amide, and carboxylic acid binding partners form a large number of reasonably stable coiled coils (T(m) > or = 60 degrees C). A detailed stability trend examination reveals that asparagine/acid pairs are sharply sensitive to acid residue chain length (Asn/Asp much worse than Asn/Glu), while guanidine/acid pairs are largely insensitive. This has been exploited to create orthogonal recognition pairs which establish the capacity to form two distinct heterodimeric coiled coils by simple mixing of four different peptides. One dimer has buried core asparagines, while the other pairs aspartic acid with any of three guanidinylated side chains. Specificity of this behavior is underscored by failure of glutamic acid substituted sequences to perform accordingly. The successful alternate pairs are further characterized by various biophysical methods (circular dichroism, ultracentrifugation, thermal and chemical denaturation, affinity tags).

Invasive plants are believed to succeed in part by secretion of allelochemicals, thus displacing competing plant species. Centaurea maculosa (spotted knapweed) provides a classic example of this process. We have previously reported that spotted knapweed roots secrete (+/-)-catechin and that (-)-catechin, but not (+)-catechin, is phytotoxic and hence may be a major contributor to C. maculosa's invasive behavior in the rhizosphere. In this communication, we explore both structure/activity relationships for flavonoid phytotoxicity and possible biosynthetic pathways for root production of (+/-)-catechin. Kaempferol and dihydroquercetin were shown to be phytotoxic, while quercetin was not. Kaempferol was converted to dihydroquercetin and (+/-)-catechin when treated with total root protein extracts from C. maculosa, but quercetin was not. This finding suggests an alteration in the standard flavonoid biosynthetic pathway in C. maculosa roots, whereby kaempferol is not a dead-end product but serves as a precursor to dihydroquercetin, which in turn leads to (+/-)-catechin production.

The design of variable-stability coiled-coil heterodimers is described. The electrostatic interface between helices, formed by contact between side chains in heptad e/g positions, is manipulated to produce complexes ranging in stability from ones that are essentially unstructured to those that cannot be thermally denatured. The tuning is accomplished by incremental extension or contraction of parent glutamic acid and lysine side chains by single methylene units, producing peptides that bear either carboxylic acids or amines separated from the peptide backbone by one to four CH2 groups. Detailed examination of all homodimers and electrostatically compatible heterodimers generates interesting combinations, particularly those in which longer-chain acids are incorporated into peptides paired with lysine-bearing ones. The discovery of very stable dimers allows exchange experiments in which one strand of an original heterodimer is specifically replaced by an added one, even in cases where the original complex features the native-like glutamic acid/lysine pairing. The reported results add significantly to the available design templates for coiled-coil construction and enable the future implementation of various triggered-recognition strategies.