Donor strand complementation mechanism in the biogenesis of non-pilus systems.

The F1 antigen of Yersinia pestis belongs to a class of non-pilus adhesins assembled via a classical chaperone-usher pathway. Such pathways consist of PapD-like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone-subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three independent methods showed that these complexes are rod- or coil-shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N-terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone-subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule-like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone-usher pathways.

Discovery of potent and selective phenylalanine derived CCR3 receptor antagonists. Part 2.

Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists.

Bacterial adhesins: structural studies reveal chaperone function and pilus biogenesis.

During the past year, remarkable progress has been made in understanding how periplasmic chaperones fold and protect protein modules that are destined for assembly into adhesive pili in Gram-negative bacteria. The first two three-dimensional structures of complexes of periplasmic chaperones with substrate pilus subunits have revealed much about the structural basis for chaperone-mediated folding and aggregation prevention, and have provided insight into the structure of adhesive pili.

Chaperone-assisted pilus assembly and bacterial attachment.

Bacterial pili assembled by the chaperone-usher pathway can mediate microbial attachment, an early step in the establishment of an infection, by binding specifically to sugars present in host tissues. Recent work has begun to reveal the structural basis both of chaperone function in the biogenesis of these pili and of bacterial attachment.

PapD-like chaperones and pilus biogenesis.

The assembly of adhesive pili from individual subunits by periplasmic PapD-like chaperones in Gram-negative bacteria offers insight into the complex process of organelle biogenesis. PapD-like chaperones bind, stabilize, and cap interactive surfaces of subunits until they are assembled into the pilus. Subunits lack the seventh *gb-strand necessary to complete their immunoglobulin-like folds; the chaperone supplies this missing strand. Indeed, the chaperone may act as a template, providing steric information to facilitate subunit folding. In the mature pilus, each subunit is thought to supply the missing strand to complete the fold of its neighbor. Thus, one general function of chaperones in organelle biogenesis may be to cap highly interactive surfaces of subunits until they reach the proper assembly site.

RSPS version 4.0: a semi-interactive vector-search program for solving heavy-atom derivatives.

A program for inspection and interpretation of the Patterson function is described. The program is mainly intended for finding heavy-atom positions from difference Patterson maps, but may also be used to locate molecules with non-crystallographic symmetry when the local axis is nearly parallel to a crystallographic symmetry axis. Options are available for vector-based methods to locate heavy-atom sites, for finding sets from a list of possible heavy-atom positions and for checking of potential solutions. Both crystallographic and non-crystallographic symmetry may be used, either independently or in conjunction.

X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli.

Type 1 pili-adhesive fibers expressed in most members of the Enterobacteriaceae family-mediate binding to mannose receptors on host cells through the FimH adhesin. Pilus biogenesis proceeds by way of the chaperone/usher pathway. The x-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli at 2.5 angstrom resolution reveals the basis for carbohydrate recognition and for pilus assembly. The carboxyl-terminal pilin domain of FimH has an immunoglobulin-like fold, except that the seventh strand is missing, leaving part of the hydrophobic core exposed. A donor strand complementation mechanism in which the chaperone donates a strand to complete the pilin domain explains the basis for both chaperone function and pilus biogenesis.

Structural basis of chaperone self-capping in P pilus biogenesis.

PapD is an immunoglobulin-like chaperone that mediates the assembly of P pili in uropathogenic strains of Escherichia coli. It binds and caps interactive surfaces on pilus subunits to prevent their premature associations in the periplasm. We elucidated the structural basis of a mechanism whereby PapD also interacts with itself, capping its own subunit binding surface. Crystal structures of dimeric forms of PapD revealed that this self-capping mechanism involves a rearrangement and ordering of the C2-D2 and F1-G1 loops upon dimerization which might ensure that a stable dimer is not formed in solution in spite of a relatively large dimer interface. An analysis of site directed mutations revealed that chaperone dimerization requires the same surface that is otherwise used to bind subunits.

Probing conserved surfaces on PapD.

PapD is the periplasmic chaperone required for the assembly of P pili in pyelonephritic strains of Escherichia coli. It consists of two immunoglobulin-like domains bisected by a subunit binding cleft. PapD is the prototype member of a super family of immunoglobulin-like chaperones that work in concert with their respective ushers to assemble a plethora of adhesive organelles including pilus- and non-pilus-associated adhesins. Three highly conserved residue clusters have been shown to play critical roles in the structure and function of PapD, as determined by site-directed mutagenesis. The in vivo stability of the chaperone depended on the formation of a buried salt bridge within the cleft. Residues along the G1 beta strand were required for efficient binding of subunits consistent with the crystal structure of PapD-peptide complexes. Finally, Thr-53, a residue that is part of a conserved band of residues located on the amino-terminal domain surface opposite the subunit binding cleft, was also found to be critical for pilus assembly, but mutations at Thr-53 did not interfere with chaperone-subunit complex formation.

Molecular basis of two subfamilies of immunoglobulin-like chaperones.

The initial encounter of a microbial pathogen with the host often involves the recognition of host receptors by different kinds of bacterial adhesive organelles called pili, fimbriae, fibrillae or afimbrial adhesins. The development of over 26 of these architecturally diverse adhesive organelles in various Gram-negative pathogens depends on periplasmic chaperones that are comprised of two immunoglobulin-like domains. All of the chaperones possess a highly conserved sheet in domain 1 and a conserved interdomain hydrogen-bonding network. Chaperone-subunit complex formation depends on the anchoring of the carboxylate group of the subunit into the conserved crevice of the chaperone cleft and the subsequent positioning of the COOH terminus of subunits along the exposed edge of the conserved sheet of the chaperone. We discovered that the chaperones can be divided into two distinct subfamilies based upon conserved structural differences that occur in the conserved sheet. Interestingly, a subdivision of the chaperones based upon whether they assemble rod-like pili or non-pilus organelles that have an atypical morphology defines the same two subgroups. The molecular dissection of the two chaperone subfamilies and the adhesive fibers that they assemble has advanced our understanding of the development of virulence-associated organelles in pathogenic bacteria.

The design and synthesis of 2,5-linked pyrrolinones. A potential non-peptide peptidomimetic scaffold.

The de novo design and initial synthetic studies directed toward construction of a novel non-peptide scaffold for beta-strand/sheet and related secondary peptide structural mimics are described. The scaffold, consisting of a repeating array of 2,5,5-trisubstituted pyrrolinone (enaminone) units punctuated with appropriate amino acid side chains, is conceptually related to our previously successful 3,5-linked polypyrrolinone non-peptide peptidomimetic scaffold. Construction of the 2,5,5-trisubstituted pyrrolinone ring system proceeds via intramolecular condensation of an N-protected amino dione. The latter is prepared from a protected alpha-amino ketone and aldehyde via an aldol-oxidation reaction sequence.

A study of independence between STR and conventional blood type loci.

Blood samples from approximately 200 Scottish Caucasian individuals were typed at conventional loci (PGM, Gc and EAP) and also with a four locus STR multiplex. Tests of the data are described which demonstrate that the assumptions of between locus independence are robust for use in forensic casework.

The use of artificial neural networks to estimate speech intelligibility from acoustic variables: a preliminary analysis.

Previous research has used regression analysis to attempt to predict the intelligibility of hearing-impaired speakers from acoustic speech parameters. Improvement of prediction may be achieved by the use of computerized artificial neural networks to process mathematically the acoustic input variables as part of the intelligibility process. A preliminary scheme for estimating speech intelligibility from acoustic parameters using a neural network is outlined and preliminary data illustrate its use.