Collagens are integral structural proteins in animal tissues and play key functional roles in cellular modulation. We sought to discover collagen model peptides (CMPs) that would form triple helices and self-assemble into supramolecular fibrils exhibiting collagen-like biological activity without preorganizing the peptide chains by covalent linkages. This challenging objective was accomplished by placing aromatic groups on the ends of a representative 30-mer CMP, (GPO)10, as with L-phenylalanine and L-pentafluorophenylalanine in 32-mer 1a. Computational studies on homologous 29-mers 1a-d (one less GPO), as pairs of triple helices interacting head-to-tail, yielded stabilization energies in the order 1a > 1b > 1c > 1d, supporting the hypothesis that hydrophobic aromatic groups can drive CMP self-assembly. Peptides 1a-d were studied comparatively relative to structural properties and ability to stimulate human platelets. Although each 32-mer formed stable triple helices (CD) spectroscopy, only 1a and 1b self-assembled into micrometer-scale fibrils. Light microscopy images for 1a depicted long collagen-like fibrils, whereas images for 1d did not. Atomic force microscopy topographical images indicated that 1a and 1b self-organize into microfibrillar species, whereas 1c and 1d do not. Peptides 1a and 1b induced the aggregation of human blood platelets with a potency similar to type I collagen, whereas 1c was much less effective, and 1d was inactive (EC50 potency: 1a/1b Ͼ Ͼ 1c > 1d). Thus, 1a and 1b spontaneously self-assemble into thrombogenic collagen-mimetic materials because of hydrophobic aromatic interactions provided by the special end-groups. These findings have important implications for the design of biofunctional CMPs.biomaterial ͉ platelets ͉ structure-function ͉ supramolecular triplex T he self-association of peptides and proteins into well ordered supramolecular structures is of pivotal importance in normal physiology and pathophysiology, such as in the assembly of collagen fibrils (1), actin filaments (2), and amyloid fibrils (3, 4). Collagens, which constitute a ubiquitous protein family in animals, contribute an essential matrix component to soft tissues and bones (5, 6). A structural hallmark of many collagens is a rope-like triple helix, the architecture of which derives from the interplay of three proline-rich polypeptide strands (e.g., two ␣1 and one ␣2 for type I collagen) (6-8). In the core domain of the triple helix, the amino acid sequence G-X-Y is repeated multiple times, and each glycine amide NH forms a hydrogen bond with the X-position amide carbonyl on an adjacent strand. The X-and Y-positions are often populated by L-proline and 4(R)-hydroxy-L-proline (O; Hyp), respectively, with the latter stabilizing the triple helix by stereoelectronic effects (9) and water-bridged hydrogen bonds (10).To investigate collagen's structure and function, researchers have resorted to using synthetic collagen model peptides (CMPs)

A Candida albicans oligopeptide transport gene, OPT1, was cloned from a C. albicans genomic library through heterologous expression in the Saccharomyces cerevisiae di-/tripeptide transport mutant PB1X-9B. When transformed with a plasmid harbouring OPT1, S. cerevisiae PB1X-9B, which did not express tetra-/pentapeptide transport activity under the conditions used, was conferred with an oligopeptide transport phenotype, as indicated by growth on the tetrapeptide Lys-Leu-Leu-Gly, sensitivity to toxic tetra- and pentapeptides, and an increase in the initial uptake rate of the radiolabeled tetrapeptide Lys-Leu-Gly-[3H]Leu. The level of oligopeptide transport was found to be influenced in the heterologous host by the source of nitrogen used for growth. The entire 3.8 kb fragment containing the oligopeptide transport activity was sequenced and an ORF of 2349 nucleotides containing a 58 nucleotide intron was identified. The deduced protein product of 783 amino acid residues contained 12 hydrophobic regions suggestive of a membrane transport protein. Sequence comparisons revealed that similar proteins are encoded by genes from S. cerevisiae and Schizosaccharomyces pombe and that OPT1 is not a member of the ABC or PTR membrane transport families.

SummaryWe have recently cloned an oligopeptide transport gene from Candida albicans denoted OPT1. This gene showed significant sequence similarity to three open reading frames (ORFs) with no previously established function: isp4 from Schizosaccharomyces pombe and Saccharomyces cerevisiae YJL212C and YPR194C, identified during the genome project. The S. pombe gene isp4 was originally identified by Sato et al. as a gene that was upregulated through nitrogen starvation induction of meiosis. However, an isp4⌬ strain exhibited a wild-type phenotype with respect to sexual differentiation. We have found that the same isp4⌬ strain is deficient in tetrapeptide transport activity as measured by its resistance to toxic tetrapeptides, by its inability to accumulate a radiolabelled tetrapeptide and by the inability to use tetrapeptides as a sole source of an amino acid to satisfy an auxotrophic requirement. Similarly, we found that the ORF YPR194C from S. cerevisiae encodes an oligopeptide transporter. Sequence analyses as well as physiological evidence has led us to propose that the proteins encoded by isp4 and the genes identified from S. cerevisiae and C. albicans comprise a new group of transporters specific for small oligopeptides, which we have named the OPT family.