Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: conformation of the bound peptide

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus. The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions. These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology. More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P. aeruginosa.     

Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy

The backbone dynamics of a ¹⁵N-labeled recombinant PAK pilin peptide spanning residues 128–144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys¹²⁸-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys¹⁴⁴) were probed by measurements of ¹⁵N NMR relaxation. This PAK(128–144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The ¹⁵N longitudinal (T₁) and transverse (T₂) relaxation rates and the steady-state heteronuclear {¹H}-¹⁵N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25°C ) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the `model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559 and 4559–4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153–162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II -turns spanning residues Asp¹³⁴-Glu-Gln-Phe¹³⁷ and Pro¹³⁹-Lys-Gly-Cys¹⁴², respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.     

Mapping the surface regions of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells

The adherence of non-mucoid Pseudomonas aeruginosa strains is believed to be mediated by the pilus, which consists of a single protein subunit of 15,000 Daltons called pilin. Ten antipeptide antisera were raised to map the surface regions of pilin from P. aeruginosa strain K (PAK). Only one of the antipeptide antisera to the eight predicted surface regions failed to react with PAK pili in direct ELISA. Five out of eight synthetic peptides representing the eight predicted surface regions reacted with anti-PAK pilus antiserum, indicating their surface exposure. Combining the antipeptide and antipilus antisera results, all eight predicted surface regions were demonstrated to be surface-exposed. The PAK 128-144-OH peptide produced the best binding antiserum to PAK pili. Only antipeptide Fab fragments directed against the disulphide bridged C-terminal region of PAK pilin blocked the adherence of pili to human buccal epithelial cells, which suggests that this region contains the receptor-binding domain of the PAK pilus.     

NMR studies of carbohydrates and carbohydrate-mimetic peptides recognized by an anti-group B Streptococcus antibody

As part of a program to investigate the origins of peptide-carbohydrate mimicry, the conformational preferences of peptides that mimic the group B streptococcal type III capsular polysaccharide have been investigated by NMR spectroscopy. Detailed studies of a dodecapeptide, FDTGAFDPDWPA, a molecular mimic of the polysaccharide antigen, and two new analogs, indicated a propensity for beta-turn formation. Different beta-turn types were found to be present in the trans and cis (Trp-10-Pro-11) isomers of the peptide: the trans isomer favored a type I beta-turn from residues Asp-7-Trp-10, whereas the cis isomer exhibited a type VI beta-turn from residues Asp-9-Ala-12. The interaction of the dodecapeptide FDTGAFDPDWPA with a protective anti-group B Streptococcus monoclonal antibody has also been investigated, by transferred nuclear Overhauser effect NMR spectroscopy and saturation-transfer difference NMR spectroscopy (STD-NMR). The peptide was found to adopt a type I beta-turn conformation on binding to the antibody; the peptide residues (Asp-7-Trp-10) forming this turn are recognized by the antibody, as demonstrated by STD-NMR experiments. STD-NMR studies of the interactions of oligosaccharide fragments of the capsular polysaccharide have also been performed and provide evidence for the existence of a conformational epitope.     

Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn

A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human buccal epithelial cell receptors of Pseudomonas aeruginosa: identification of glycoproteins with pilus binding activity

Adherence of Pseudomonas aeruginosa to a patient's epithelial surface is thought to be an important first step in the infection process. Pseudomonas aeruginosa is capable of attaching to epithelial cells via its pili, yet little is known about the epithelial receptors of this adhesin. Using nitrocellulose replicas of polyacrylamide gels of solubilized human buccal epithelial cells (BECs), glycoproteins (Mz: 82,000, and four bands between 40,000 and 50,000) that bound purified pili from P. aeruginosa strain K (PAK) were identified by immunoblotting with a pilus-specific monoclonal antibody that does not affect pilus binding to BECs (PK3B). All pilus-binding glycoproteins were surface localized, as determined by surface radioiodination of intact BECs. Binding of pili to all of the glycoproteins was inhibited by Fab fragments of monoclonal antibody PK99H, which inhibits PAK pili binding to BECs by binding to or near the binding domain of the pilus, but not by Fab fragments of monoclonal antibody PK41C, which binds to PAK pilin but does not inhibit pili binding to BECs, demonstrating that pilus binding to these glycoproteins is likely via the same region of the pilus that binds to intact BECs. Periodate oxidation of the blot eliminated pili binding to all glycoproteins, indicating that a carbohydrate moiety is an important determinant for pilus-binding activity. However, not all of the glycoproteins exhibited the same degree of sensitivity to periodate oxidation. Furthermore, monosaccharide inhibition of pilus binding to BECs implicated L-fucose and N-acetylneuraminic acid as receptor moieties.     

Antigen-antibody interactions: elucidation of the epitope and strain-specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K.

The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain-specific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-Phe-Ile-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain-specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.     

The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for both biotic and abiotic surfaces

Pseudomonas aeruginosa readily binds to stainless steel and other abiotic surfaces, causing major problems in both the medical and food industries. In this study, we show that P. aeruginosa binds to abiotic surfaces in a concentration-dependent, saturable manner during the initial stages of biofilm formation. P. aeruginosa type IV pili mediate binding to stainless steel as a pilus-deficient strain does not bind to steel, purified type IV pili bound in a concentration-dependent, saturable manner, and purified pili competitively inhibited whole cell binding. PAK pili can also bind polystyrene and polyvinylchloride in a concentration-dependant and saturable manner. As an antibody specific for the C-terminal pilin receptor binding domain inhibited adherence to abiotic surfaces, the role of the C-terminal receptor binding domain in mediating binding to steel surfaces was examined. A synthetic peptide of the PAK pilin epithelial cell receptor binding domain [PAK(128-144)ox] bound directly to steel with high affinity. The interaction of pili with steel was specifically inhibited by this peptide with an apparent Ki of approximately 0.2 nM and effectively inhibited the binding of viable homologous and heterologous P. aeruginosa strains to steel with an apparent Ki of approximately 4 nM. A single point mutation (K130I) in the PAO receptor binding domain was observed to abolish binding to stainless steel while binding to human buccal epithelial cells was enhanced. Therefore, the C-terminal receptor binding domain appears to have evolved for binding a variety of surfaces.     

Comparative studies of the amino acid and nucleotide sequences of pilin derived from Pseudomonas aeruginosa PAK and PAO.

The entire amino acid sequence for Pseudomonas aeruginosa PAO pilin was determined through peptide sequencing and from the complete nucleotide sequence encoding the pilin gene. The precursor PAO pilin is 149 amino acids in length which includes a 6-amino-acid positively charged leader sequence. Comparison of the amino acid sequences of pilin produced by P. aeruginosa PAO and PAK reveals a region of high homology corresponding to the leader peptide and residues 1 to 54 of the mature pilin. The amino acid sequence of the peptide encompassing the major antigenic determinant of PAK differs greatly from that of the equivalent region in PAO. The C-terminal regions of these proteins are semiconserved. Few major differences were found when the predicted secondary structures for PAO and PAK pilins were compared. Major nucleotide sequence variation between the equivalent restriction fragments from PAO and PAK occurred within the areas coding for the peptides containing the immunodominant site for PAK pilin and the C termini.     

Solution structure of the cyclic peptide contryphan-Vn, a Ca2+-dependent K+ channel modulator

The solution structure of contryphan-Vn, a cyclic peptide with a double cysteine S-S bridge and containing a D-tryptophan extracted from the venom of the cone snail Conus ventricosus, has been determined by NMR spectroscopy using a variety of homonuclear and heteronuclear NMR methods and restrained molecular dynamics simulations. The main conformational features of backbone contryphan-Vn are a type IV beta-turn from Gly 1 to Lys 6 and a type I beta-turn from Lys 6 to Cys 9. As already found in other contryphans, one of the two prolines--the Pro4--is mainly in the cis conformation while Pro7 is trans. A small hydrophobic region probably partly shielded from solvent constituted from the close proximity of side chains of Pro7 and Trp8 was observed together with a persistent salt bridge between Asp2 and Lys6, which has been revealed by the diagnostic observation of specific nuclear Overhauser effects. The salt bridge was used as a restraint in the molecular dynamics in vacuum but without inserting explicit electrostatic contribution in the calculations. The backbone of the unique conformational family found of contryphan-Vn superimposes well with those of contryphan-Sm and contryphan-R. This result indicates that the contryphan structural motif represents a robust and conserved molecular scaffold whose main structural determinants are the size of the intercysteine loop and the presence and location in the sequence of the D-Trp and the two Pro residues.     

Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor-binding domain

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt- P. aeruginosa ) and C. albicans whole cells (Bt- C. albicans ). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.     

DNA binding: a novel function of Pseudomonas aeruginosa type IV pili

The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.     

A cis proline turn linking two beta-hairpin strands in the solution structure of an antibody-bound HIV-1IIIB V3 peptide

The refined solution structure of an 18-residue HIV-1IIIB V3 peptide in complex with the Fv fragment of an anti-gp120 antibody reveals an unexpected type VI beta-turn comprising residues RGPG at the center of a beta-hairpin. The central glycine and proline of this turn are linked by a cis peptide bond. The residues of the turn interact extensively with the antibody Fv. 15N[1H] NOE measurements show that the backbone of the peptide, including the central QRGPGR loop, is well ordered in the complex. The solution structure is significantly different from the X-ray structures of HIV-1MN V3 peptides bound to anti-peptide antibodies. These differences could be due to a two-residue (QR) insertion preceding the GPGR sequence in the HIV-1IIIB strain, and the much longer peptide epitope immobilized by the anti-gp120 antibody.     

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.     

A cis-prolyl peptide bond isomerization dominates the folding of the alpha subunit of Trp synthase,a TIM barrel protein

The cis/trans isomerization of prolyl peptide bonds has been suggested to dominate the folding of the alpha subunit of tryptophan synthase from Escherichia coli (alphaTS). To test the role of the unique cis isomer between Asp27 and Pro28, the folding properties of P28A, P28G and G(3)P28G, a three-glycine insertion mutant between Asp27 and Gly28, were investigated using urea as a denaturant. Circular dichroism analysis demonstrated that none of the mutations perturb the secondary structure significantly, although the aromatic side-chain packing is altered for P28A and P28G. All three mutant proteins inherited the three-state thermodynamic behavior observed in wild-type alphaTS, ensuring that the fundamental features of the energy surface are intact. Kinetic studies showed that neither alanine nor glycine substitutions at Pro28 results in the elimination of any slow-refolding phases. By contrast, the G(3)P28G mutant eliminates the fastest of the slow-refolding phases and one of the two unfolding phases. Double-jump experiments on G(3)P28G confirm the assignment of the missing refolding phase to the isomerization of the Asp27-Pro28 peptide bond. These results imply that the local stability conveyed by the tight, overlapping turns containing the cis peptide bond is sufficient to favor the cis isomer for several non-prolyl residues. The free energy required to drive the isomerization reaction is provided by the formation of the stable intermediate, demonstrating that the acquisition of structure and stability is required to induce subsequent rate-limiting steps in the folding of alphaTS.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics

Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilA(PAK) pilin subunit in N. gonorrhoeae. We show here that, although PilA(PAK) pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilA(PAK) pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilA(PAK) pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilA(PAK) pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilA(PAK) pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.     

Pilin C-terminal peptide binds asialo-GM1 in liposomes: a 2H-NMR study.

Wideline 2H-NMR observations are described demonstrating the interaction of a synthetic peptide (PAK), representing residues 128-144 of the binding domain of pilin surface protein from Pseudomonas aeruginosa, with a complex glycosphingolipid thought to be its natural receptor. The receptor glycolipid (asialo-GM1) carried 2H probe nuclei on the terminal and next-to-terminal carbohydrate residues and was present as a minor component in fluid phosphatidylcholine liposomes. The peptide induced spectral changes that could be understood as arising from receptor motional changes, without receptor immobilization on the NMR time scale of 10(4) s-1. Spectral effects were reversed by reduction of the single peptide disulfide bond--a structural feature previously shown to be a determinant of PAK conformation (Campbell AP, McInnes C, Hodges RS, Sykes BD. 1995. Biochemistry 34:16255-16268). This is the first demonstration of PAK interaction with its epithelial cell receptor in liposomes.     

The expression of mutant pilins in Pseudomonas aeruginosa: fifth position glutamate affects pilin methylation

The expression within Pseudomonas aeruginosa PAO1 of three mutant pilin genes from P. aeruginosa PAK was studied to determine their effects on pilin stability, translocation into the membrane, leader peptide removal, and methylation of the mature N-terminal phenylalanine. The results revealed that a deletion of 4 or 8 amino acids within the immediate N-terminus of pilin had deleterious effects upon leader peptide cleavage. In addition, while the 4-amino-acid deletion did not affect pilin partitioning into the membrane, the 8-amino-acid deletion decreased the amount of pilin found within the membrane fraction. Of considerable interest was the finding that the mutation within the mature pilin of the glutamate at position 5 to a lysine did not prevent leader peptide removal but did inhibit the methylation of the N-terminal phenylalanine.     

Configurationally driven folding of model tetrapeptides containing L- or D-morpholine-3-carboxylic acids as beta-turn nucleators

The conformational analysis by NMR, IR, and molecular modeling of tetrapeptides containing morpholine-3-carboxylic acid (Mor) as a proline surrogate is presented. The relationship between the chirality of the cyclic amino acid at position i+1 and the turn propensity is maintained with respect to the reference proline-containing peptides, although marked differences in the type of folded structures were observed. The conformational profile of morpholine-containing turn peptides as a function of the chirality of the cyclic amino acid indicated that the heterochiral tetrapeptide containing the D-isomer of the cyclic amino acid is more prone to nucleate compact folded structures, although with no resemblance to the beta-turn structures of D-proline-containing peptides. Also, the solvation system proved to influence the organization of folded structures, as in the more interactive CD(3)CN the model peptides showed more compact conformations. The L-Mor-containing peptide displayed two rotamers at the Val-Mor amide bond. The trans isomer did not experience any turn structures, nor any intramolecular hydrogen-bonds, whereas the cis isomer showed a strong preference for a type VI beta-turn structure, thus providing a different conformational asset with respect to the beta-turn structure as reported for the reference L-proline model peptide.