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R S Hodges

Publications and source records attributed to R S Hodges.

At least 73 records · Page 4Linked to original sources
PubMed1998

Effects of lanthanide binding on the stability of de novo designed alpha-helical coiled-coils.

Effects of La3+ ion binding on the stability of de novo designed two-stranded alpha-helical coiled-coils were studied. The coiled-coils were composed of two 35-residue polypeptide chains based on the "native" heptad sequence Q(g)V(a)G(b)A(c)L(d)Q(e)K(f) and each contained a Cys residue at position 2a to allow formation of an interchain disulfide bridge. The effect of LaCl3 on the stability of five analogs containing two or three Glu substitutions per chain at heptad positions e and g was observed by urea denaturation at 20 degrees C. The analog E2(15,20), in which Glu residues are involved in interhelical i to i' + 5 repulsions, was stabilized relative to the control native peptide by addition of 50 mM LaCl3 to the buffer, whereas two analogs, in which Glu residues do not interact, were destabilized. These results suggest that LaCl3 may preferentially stabilize the folded state of E2(15,20) by the "bridging" of La3+ ions between two pairs of Glu residues usually involved in interhelical repulsions. Two analogs designed to contain two La3+ binding sites composed of three Glu residues each show greater stabilization by LaCl3 than E2(15,20) in the disulfide-bridged form. The apparent stabilization of E2(15,20) by La3+ binding was not observed with either Ca2+ or Mg2+, indicating that the effect is specific for trivalent versus divalent cations.

Amino Acid Sequence↗
PubMed1998

The use of synthetic peptides in the design of a consensus sequence vaccine for Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. Research has shown that the C-terminal region of the pilin monomer contains the epithelial cell binding domain, which is semiconserved in seven different strains of this bacterium. Antibodies to this region of the pilin molecule are also able to block and prevent the infection process. As there is a degree of sequence and structural homology in the C-terminal region and all strains examined have been shown to bind to the same cell surface receptor, we reasoned that it should be possible to produce a synthetic peptide consensus sequence which would provide cross-reactive antiserum from a single peptide immunogen inhibiting the adherence of the known strains of P. aeruginosa. In this article we examine the cross-reactivity of five rabbit polyclonal antisera. One has been raised against the cell-surface receptor binding domain of native PAK strain pilin (residues 128-144) while the others have been raised to analogues of this region. Analysis of the cross-reactivity of these antisera, using competitive ELISA assay, has shown that it is possible to manipulate the amino acid sequence of a peptide immunogen to generate antiserum, which exhibits enhanced cross-reactivity to various strains of P. aeruginosa. Furthermore, when this peptide is conjugated to tetanus toxoid and used to vaccinate mice it provided cross-reactive protection against heterologous challenge with PAO strain bacteria. The results of these experiments are analyzed, and the applicability of our hypothesis and the implications of this approach to the design of a strain-independent consensus vaccine for immunization against Pseudomonas aeruginosa are discussed.

Amino Acid Sequence↗
PubMed1998

Influence of preformed alpha-helix and alpha-helix induction on the activity of cationic antimicrobial peptides.

One prominent class of cationic antibacterial peptides comprises the alpha-helical class, which is unstructured in free solution but folds into an amphipathic alpha-helix upon insertion into the membranes of target cells. To investigate the importance of alpha-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form alpha-helices or to have preformed alpha-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i + 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized alpha-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of alpha-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase alpha-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r2 = 0.88) with a helican parameter which was calculated as the induction of alpha-helix in a membrane-mimicking environment divided by the alpha-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.

Amino Acid Sequence↗
PubMed1997

Effect of mobile phase on the oligomerization state of alpha-helical coiled-coil peptides during high-performance size-exclusion chromatography.

Important structural motifs involving amphipathic helices include two-stranded and multiple-stranded coiled-coils. High-performance size-exclusion chromatography (HPSEC) is a useful tool to examine both the oligomerization state of coiled-coils as well as the stability of such motifs, due to the facile manipulation of the mobile phase and the lack of interaction of the peptide solutes with the stationary phase. In the present study, HPSEC was applied to two series of de novo designed model amphipathic alpha-helical peptides with the sequences (1) Ac-(E-A-L-K-A-E-I)n-E-A-C-K-A-amide, where n = 1 or 3, Ac-E-I-(E-A-L-K-A-E-I)4-E-A-C-K-A-amide and (2) Ac-(K-L-E-A-L-E-A)n-amide, where n = 1, 2 or 4. Observation of the retention behaviour of Series 1 under both denaturing and non-denaturing conditions at pH 7.0 offered insights into the effect of polypeptide chain length and disulphide bridge formation on the stability of alpha-helical coiled-coils. In contrast, the Series 2 peptides showed promise as peptide standards to monitor the effect of environment on the multi-strandedness of coiled-coils, since the 28-residue peptide of this series was eluted as a monomer, dimer or trimer depending on mobile phase conditions.

Amino Acid Sequence↗
PubMed1997

A diminished role for hydrogen bonds in antifreeze protein binding to ice.

The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP1) in winter flounder is a 37-amino-acid-long, alanine-rich, alpha-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {2021} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice.

Amino Acid Sequence↗
PubMed1997

Interaction of the second binding region of troponin I with the regulatory domain of skeletal muscle troponin C as determined by NMR spectroscopy.

Two dimensional 1H,15N-heteronuclear single quantum correlation NMR was used to monitor the resonance frequency changes of the backbone amide groups belonging to the 15N-labeled regulatory domain of calcium saturated troponin C (N-TnC) upon addition of synthetic skeletal N-acetyl-troponin I 115-131-amide peptide (TnI115-131). Utilizing the change in amide chemical shifts, the dissociation constant for 1:1 binding of TnI115-131 to N-TnC in low salt and 100 mM KCl samples was determined to be 28 +/- 4 and 24 +/- 4 microM, respectively. The off rate of TnI115-131 was determined to be 300 s-1 from observed N-TnC backbone amide 1H,15N-heteronuclear single quantum correlation cross-peak line widths, which is on the order of the calcium off rates (Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry 34, 8330-8340), and agrees with kinetic expectations for biological regulation of muscle contraction. The TnI115-131 binding site on N-TnC was determined by mapping of chemical shift changes onto the N-TnC NMR structure and was demonstrated to be in the "hydrophobic pocket" (Gagné, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Nat. Struct. Biol. 2, 784-789).

Animals↗
PubMed1997

Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study.

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has recently been the target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. We have successfully cloned and bacterially expressed a 15N-labeled PAK pilin peptide spanning residues 128-144 of the intact PAK pilin protein, PAK 128-144(Hs145), and have determined the solution secondary structure of this peptide using heteronuclear multidimensional NMR spectroscopy. The oxidized recombinant peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around the Ile138-Pro139 peptide bond. The pattern of NOEs, temperature coefficients, and coupling constants observed for the trans isomer demonstrate the presence of a type I beta-turn and a type II beta-turn spanning Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively. This is in agreement with the NMR solution structure of the trans isomer of a synthetic PAK 128-144 peptide which showed a type I and a type II beta-turn in these same regions of the sequence [McInnes, C., Sönnichsen, F. D., Kay, C. M., Hodges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hodges, R. S., and Sykes, B. D. (1995) Biochemistry 34, 16255-16268]. The pattern of NOEs, temperature coefficients, and coupling constants observed for the cis isomer also demonstrate a type II beta-turn spanning Pro139-Lys-Gly-Cys142, but suggest a second beta-turn spanning Asp132-Gln-Asp-Glu135. Thus, the cis isomer may also possess a double-turn motif (like the trans isomer), but with different spacing between the turns and a different placement of the first turn in the sequence. The discovery of a double-turn motif in the trans (and cis) recombinant PAK pilin peptide is an extremely important result since the double turn has been implicated as a structural requirement for the recognition of both receptor and antibody. These results pave the way for future isotope-edited NMR studies of the labeled recombinant PAK pilin peptide bound to antibody and receptor, studies integral to the design of an effective synthetic peptide vaccine.

Amino Acid Sequence↗
PubMed1997

Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction.

To investigate the functional importance of the C-terminal residues 116 to 148 of troponin I (TnI) in the Ca2+-dependent regulation of vertebrate skeletal muscle contraction, we have prepared several synthetic TnI peptide analogs corresponding to various regions within residues 96 to 148 of rabbit skeletal TnI, and analyzed each of these peptides in reconstituted thin filament assays. Our results show that the TnI peptide 96 to 148 (TnI96-148) constitutes the minimal sequence of TnI capable of mediating an inhibitory activity similar to that of intact TnI protein. Truncation of residues 140 to 148 from this region (TnI96-139) or substitution of residues K141, K142 and K144 with alanine (TnI96-148A2) completely abolishes the enhanced inhibitory effect of this region when compared with TnI96-115. A synthetic peptide, residues 128 to 148 of TnI, containing residues 140 to 148, now termed the "second actin-tropomyosin (actin-Tm) binding site" is able to bind specifically to the actin-Tm filament and can induce a weak inhibitory activity on its own. Residues 116 to 131 of TnI do not appear to be important for inhibition, but are critical for interacting with troponin C (TnC). Specific investigations into this region have shown that residues 116 to 126, located directly adjacent to the "inhibitory region" (residues 96 to 115), are critical for allowing TnC to neutralize fully and rapidly the acto-S1-Tm inhibition caused by the various TnI peptides. Furthermore, residues 116 to 131 of TnI, now termed the "second TnC binding site", can significantly enhance the binding affinity of the inhibitory region, residues 96 to 115, for TnC in a Ca2+-dependent manner as determined by affinity chromatography analysis. The implication that TnI residues 116 to 131 bind to the N domain of TnC, and thus the inhibitory region (residues 96 to 115) binds to the C domain of TnC, has made us re-investigate the structural/functional role of the NH2-terminal region of TnI. Studies of competition between the N terminus of TnI (Rp1-40, residues 1 to 40) with the C-terminal peptides TnI96-115, TnI96-131 and TnI96-148 showed that only TnI96-115 could be easily displaced from TnC. These results thus suggest that Ca2+ binding to the regulatory sites of TnC (N domain) alters the binding affinity between the NH2 terminus and the C terminus of TnI for TnC, i.e. a Ca2+-dependent switch between these two sites of TnI for the C domain of TnC. These results have been incorporated into a general model describing the Ca2+-dependent regulation of muscle contraction.

Actins↗
PubMed1997

Use of sodium perchlorate at low pH for peptide separations by reversed-phase liquid chromatography. Influence of perchlorate ion on apparent hydrophilicity of positively charged amino acid side-chains.

The reversed-phase liquid chromatography (RPLC) behavior of synthetic model peptides containing positively charged amino acid residues was studied in the presence or absence of 100 mM sodium perchlorate in order to determine the effect on apparent side-chain hydrophilicity of a charged residue at low pH. The peptides used in this study were either non-helical peptides or amphipathic alpha-helical peptides, where the effect of the negatively charged perchlorate ion on a charged residue in either the hydrophobic face or hydrophilic face of the helix was monitored. We have shown that the addition of 100 mM perchlorate to RPLC separations of positively charged peptides performed in a 20 mM aqueous phosphoric acid-acetonitrile system resulted in an increase in retention time of a peptide relative to the same peptide in the absence of perchlorate. This effect occurred independent of conformation, i.e., whether comparing the effect of positively charged residue substitutions in the hydrophobic or hydrophilic face of an amphipathic alpha-helix or in a peptide with negligible secondary structure. From these results, suggesting that positively charged side-chain hydrophilicity is decreased by ion-pairing with the perchlorate ion, we have shown practical examples where mixtures of non-helical and amphipathic alpha-helical peptides showed enhanced resolution in the presence of perchlorate at pH 2, compared to in its absence. In addition, it was shown that an aqueous phosphoric acid-perchlorate-acetonitrile mobile phase may show markedly different selectivity for peptide separations at low pH compared to the more traditional aqueous trifluoroacetic acid-acetonitrile system.

Amino Acid Sequence↗
PubMed1997

Nonlamellar phases induced by the interaction of gramicidin S with lipid bilayers. A possible relationship to membrane-disrupting activity.

The interactions of the cyclic peptide gramicidin S (GS) with a variety of single-component lipid bilayers, and with membrane polar lipid extracts of Acholeplasma laidlawii B and Escherichia coli, were examined by differential scanning calorimetry (DSC), 31P-nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction. The DSC data indicate that the effects of GS on the thermotropic phase behavior of phosphatidylcholine and phosphatidylethanolamine dispersions are compatible with those expected of peptides interacting primarily with the polar headgroup and/or the polar/apolar interfaces of lipid bilayers. These DSC studies also suggest that GS exhibits stronger interactions with the more fluid bilayers. For mixtures of GS with lipids such as phosphatidylcholine, phosphatidylserine, cardiolipin, and sphingomyelin, axially symmetric 31P-NMR powder patterns are observed throughout the entire temperature range examined (0-90 degrees C), and there is little evidence for significant destabilization of the lipid bilayer with respect to nonlamellar phases. With mixtures of GS with either phosphatidylethanolamine, phosphatidylglycerol, or a nonlamellar phase-forming phosphatidylcholine, axially symmetric 31P-NMR powder patterns are also observed at low temperatures. However, at high temperatures, an isotropic component is observed in their 31P-NMR spectra, and the relative intensity of this component increases significantly with temperature and with GS concentration. Once formed at high temperatures, this isotropic component exhibits a marked cooling hysteresis and in most cases disappears only when the sample is recooled to temperatures well below the lipid hydrocarbon chain-melting phase transition temperature. We also show that GS induces the formation of isotropic components in the 31P-NMR spectra of heterogeneous lipid mixtures such as occur in A. laidlawii B and E. coli membranes. These observations suggest that GS induces the formation of cubic or other three dimensionally ordered inverted nonlamellar phases when it interacts with some types of lipid bilayers, a suggestion strongly supported by our X-ray diffraction studies. Our results also suggest that the capacity of GS to induce the formation of such phases increases with the intrinsic nonlamellar phase-preferring tendencies of the lipids with which it interacts probably by producing localized increases in membrane monolayer curvature stress. The latter effect could be part of the mechanism through which this peptide exhibits its antimicrobial and hemolytic activities.

Calorimetry, Differential Scanning↗
PubMed1997

DnaJ potentiates the interaction between DnaK and alpha-helical peptides.

Molecular chaperones bind selectively to nascent, unfolded, misfolded, or aggregated polypeptides, and are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. The DnaK chaperone of Escherichia coli is known to interact preferentially with positively charged hydrophobic peptides in an extended conformation. Accordingly, we show in the present study that DnaK has a low affinity for alpha-helical peptides. In the presence of its co-chaperone DnaJ and ATP, however, DnaK interacts more efficiently with alpha-helical peptides. This suggests that DnaJ triggers a conformational change in DnaK which improves its interaction with these peptides. The ability of the DnaK/DnaJ/GrpE chaperone machine to interact with alpha-helical peptides (which represent the most frequent secondary structure in proteins) should be an important part of its role in protein folding and renaturation.

Adenosine Triphosphate↗
PubMed1997

Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament.

The regions of troponin I (TnI) responsible for Ca2+-dependent activation and Ca2+ sensitivity of the actin-myosin subfragment 1-tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATPase assay at pH 7.8 has been applied to reconstituted skeletal muscle thin filaments at actin:S1:TM ratios of 6:1:2. Several TnI fragments (TnI-(104-115), TnI-(1-116), and TnI-(96-148)) and TnI mutants with single amino acid substitutions within the inhibitory region (residues 104-115) were assayed to determine their roles on the regulatory function of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly shown by the reduced potency of TnI mutants with single amino acid substitutions within this region. However, the function of the inhibitory region is modulated by other regions of TnI as observed by the poor inhibitory activity of TnI-(1-116) and the increased potency of the inhibitory region by TnI-(96-148). The regulatory complex composed of TnI-(96-148) plus troponin T-troponin C complex (TnT.C) displays the same Ca2+ sensitivity (pCa50) as intact troponin (Tn) or TnI plus TnT.C while those regulatory complexes composed of TnT.C plus either TnI-(104-115) or TnI-(1-116) had an increase in their pCa50 values. This indicates that the Ca2+ sensitivity or responsiveness of the thin filament is controlled by TnI residues 96-148. The ability of Tn to activate the acto-S1-TM ATPase activity in the presence of calcium to the level of the acto-S1 rate was mimicked by the regulatory complex composed of TnI-(1-116) plus TnT.C and was not seen with complexes composed with either TnI-(104-115) or TnI-(96-148). This indicates that the N terminus of TnI in conjunction with TnT controls the degree of activation of the ATPase activity. Although the TnI inhibitory region (104-115) is the Ca2+-sensitive switch which changes binding sites from actin-TM to TnC in the presence of calcium, its function is modulated by both the C-terminal and N-terminal regions of TnI. Thus, distinct regions of TnI control different aspects of Tn's biological function.

Actin Cytoskeleton↗
PubMed1997

Salt effects on protein stability: two-stranded alpha-helical coiled-coils containing inter- or intrahelical ion pairs.

An investigation into the role of surface-accessible ion pairs in protein stability was carried out by determining the effects of added salt (KCl, MgCl2 and LaCl3) at neutral and acidic pH on the stability of de novo designed two-stranded alpha-helical coiled-coils. The effects of salt on the stability of coiled-coils containing interhelical i to i' + 5 or intrahelical i to i + 4 and i to i + 3 Lys-Glu ion pairs were compared to the effects on the stability of a control coiled-coil, which contained no intra- or interhelical ion pair. These studies indicate that ionic interactions contribute to coiled-coil stability. The results show that added salt can have complex effects on protein stability, involving stabilizing and destabilizing contributions with the net effect depending on the nature of the charged residues and ionic interactions present in the protein.

Amino Acid Sequence↗
PubMed1997

Demonstration of coiled-coil interactions within the kinesin neck region using synthetic peptides. Implications for motor activity.

Kinesin is a dimeric motor protein that can move for several micrometers along a microtubule without dissociating. The two kinesin motor domains are thought to move processively by operating in a hand-over-hand manner, although the mechanism of such cooperativity is unknown. Recently, a approximately 50-amino acid region adjacent to the globular motor domain (termed the neck) has been shown to be sufficient for conferring dimerization and processive movement. Based upon its amino acid sequence, the neck is proposed to dimerize through a coiled-coil interaction. To determine the accuracy of this prediction and to investigate the possible function of the neck region in motor activity, we have prepared a series of synthetic peptides corresponding to different regions of the human kinesin neck (residues 316-383) and analyzed each peptide for its respective secondary structure content and stability. Results of our study show that a peptide containing residues 330-369 displays all of the characteristics of a stable, two-stranded alpha-helical coiled-coil. On the other hand, the NH2-terminal segment of the neck (residues approximately 316-330) has the capacity to adopt a beta-sheet secondary structure. The COOH-terminal residues of the neck region (residues 370-383) are not alpha-helical, nor do they contribute significantly to the overall stability of the coiled-coil, suggesting that these residues mark the beginning of a hinge located between the neck and the extended alpha-helical coiled coil stalk domain. Interestingly, the two central heptads of the coiled-coil segment in the neck contain conserved, "non-ideal" residues located within the hydrophobic core, which we show destabilize the coiled-coil interaction. These residues may enable a portion of the coiled-coil to unwind during the mechanochemical cycle, and we present a model in which such a phenomenon plays an important role in kinesin motility.

Amino Acid Sequence↗
PubMed1997

Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design.

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems.

Amino Acid Sequence↗
PubMed1997

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.

Amino Acid Sequence↗