Annett Rozek - Academia.edu (original) (raw)

Papers by Annett Rozek

Research paper thumbnail of The role of structure in antibody cross-reactivity between peptides and folded proteins 1 1 Edited by J. A. Wells

Journal of Molecular Biology, 1998

Peptides have the potential for targeting vaccines against pre-speci®ed epitopes on folded protei... more Peptides have the potential for targeting vaccines against pre-speci®ed epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced ®t of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with submicromolar af®nities. The strong preference for a type II b-turn predicted for one peptide was con®rmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.

Research paper thumbnail of The role of structure in antibody cross-reactivity between peptides and folded proteins1

Journal of Molecular Biology, 1998

Peptides have the potential for targeting vaccines against pre-speci®ed epitopes on folded protei... more Peptides have the potential for targeting vaccines against pre-speci®ed epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced ®t of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with submicromolar af®nities. The strong preference for a type II b-turn predicted for one peptide was con®rmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.

Research paper thumbnail of Sequence-specific 1 H NMR resonance assignments and secondary structure of human apolipoprotein CI in the presence of sodium dodecyl sulfate

Biochemistry and Cell Biology-biochimie Et Biologie Cellulaire, 1998

Apolipoprotein (apo) C-I is a 57-residue exchangeable plasma protein distributed mainly in high a... more Apolipoprotein (apo) C-I is a 57-residue exchangeable plasma protein distributed mainly in high and very low density lipoprotein. In this report we present the nuclear magnetic resonance spectra of native apoC-I and synthetic apoC-I, containing selected 15N-labelled amino acids, in the presence of sodium dodecyl sulfate. The proton resonances of apoC-I are assigned and the secondary structure is estimated from the difference of measured alpha-proton chemical shifts to random coil values and the observed NOE interactions. According to these data apoC-I forms two helices, Val-4-Lys-30 and Leu-34-Lys-52, linked by an unstructured region Gln-31-Glu-33. The N-terminal segments of each helix, Val-4-Gly-15 and Leu-34-Met-38, appear to be more flexible than the helical core regions Asn-16-Lys-30 and Arg-39-Lys-52.

Research paper thumbnail of Infrared Spectroscopy of Human Apolipoprotein Fragments in SDS/D 2 O: Relative Lipid-Binding Affinities and a Novel Amide I Assignment

Biochemistry, 1997

Infrared absorption spectra are reported for six apolipoprotein fragments in SDS/D 2 O. Five of t... more Infrared absorption spectra are reported for six apolipoprotein fragments in SDS/D 2 O. Five of the peptides correspond to proposed lipid-binding domains of human apolipoproteins [apoC-I(7-24), apoC-I(35-53), apoA-II(18-30)+, apoA-I(166-185), apoE(267-289)], and the sixth is the de noVo lipid associating peptide LAP-20. The amide I infrared absorption patterns are generally consistent with predominantly helical structures (as determined previously by NMR spectroscopy and distance geometry calculations) and further suggest that apoA-I(166-185) and apoE(267-289) are bound to SDS relatively weakly in comparison to the other four peptides. The latter conclusion is also supported by the temperature dependence of the infrared spectra, as increasing temperature promotes a distinct increase in random coil structure only for apoA-I(166-185) and apoE . In addition to features readily ascribed to helices, the infrared spectra of all the peptides show absorptions in the spectral region 1630-1635 cm -1 that is usually associated with -structure, a motif that is clearly absent from the NMR-derived structures. Parallel difficulties also arose in the analyses of the circular dichroism spectra. We suggest that both the lowfrequency infrared absorptions and the ambiguities in interpreting the CD spectra may be due to unusual structures at the peptide C-termini, involving CdO groups that form hydrogen bonds simultaneously either with two solvent molecules or with donors from the backbone (NH) and the solvent (OH). Analogous absorptions may be a general feature of solvent-exposed helices, which suggests a need for caution in assigning amide I bands below 1640 cm -1 . † Issued as NRCC Publication No. 34783.

Research paper thumbnail of Structural studies of a baboon ( Papio sp. ) plasma protein inhibitor of cholesteryl ester transferase

Protein Science, 2000

A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in... more A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N-terminus of baboon apoC-I. Relative to CETIP, baboon apoC-I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid-phase methods. Using sodium dodecyl sulfate (SDS) to model the lipoprotein environment, the solution structure of CETIP was probed by optical and 1HNMR spectroscopy. Circular dichroism data show that the protein lacks a well-defined structure in water but, upon the addition of SDS, becomes helical (56%). A small blue shift of 8 nm was observed in the intrinsic tryptophan fluorescence of CETIP in the presence of saturating amounts of SDS, suggesting that tryptophan-23 is not buried deeply in the lipid environment. The helical nature of CETIP in the presence of SDS was confirmed by upfield 1Hα secondary shifts and an average solution structure determined by distance geometry/simulated annealing calculations using 476 NOE-based distance restraints. The backbone (N — Cα — Cα = O ) root-mean-square deviation of an ensemble of 17 out of 25 calculated structures superimposed on the average structure was 1.06 ± 0.30 Å using residues V4-P35 and 0.51 ± 0.17 Å using residues A7-S32. Although the side-chain orientations fit the basic description of a class A amphipathic helix, both intramolecular salt bridge formation and “snorkeling” of basic side chains toward the polar face play minor, if any, roles in stabilizing the lipid-bound amphipathic structure. Conformational features of the calculated structures for CETIP are discussed relative to models of CETIP inhibition of cholesteryl ester transferase.

Research paper thumbnail of Conformation of Two Peptides Corresponding to Human Apolipoprotein CI Residues 7-24 and 35-53 in the Presence of Sodium Dodecyl Sulfate by CD and NMR Spectroscopy

Biochemistry, 1995

Peptides corresponding to the proposed lipid-binding domains of human apolipoprotein C-I, residue... more Peptides corresponding to the proposed lipid-binding domains of human apolipoprotein C-I, residues 7-24 (ALDKLKEFGNTLEDKARE) and 35-53 (SAKMREWFSETFQKVKEKL), were studied by CD and two-dimensional 'H NMR spectroscopy. Sodium dodecyl sulfate (SDS) was used to model the lipoprotein environment. Analysis of the CD data shows that both peptides lack well-defined structure in aqueous solution but adopt helical, ordered structures upon the addition of SDS. The helical nature of the peptides in the presence of SDS was confirmed by Ha secondary shifts. A total of 199 (apoC-1(7-24)) and 266 (apoC-I(35-53)) distance restraints were used in distance geometry and simulated annealing calculations to generate average structures for both peptides in aqueous solutions containing SDS. The backbone (N, Ca, C=O) RMSD from the average structure of an ensemble of 20 structures was 0.73 f 0.22 and 0.48 f 0.14 A for apoC-I(7-24) and apoC-1(35-53), respectively. In the presence of SDS, the distance geometry and simulated annealing calculations show that both peptides adopt well-defined amphipathic helices with distinct hydrophobic and hydrophilic faces. The calculated structures are discussed relative to predicted structures. Comparing our CD and NMR results for the apoC-I fragments in SDS with CD results of others obtained in the presence of dimyristoylphosphatidylcholine indicates that SDS may be a better model of the lipoprotein environment.

Research paper thumbnail of Structure of a biologically active fragment of human serum apolipoprotein C-II in the presence of sodium dodecyl sulfate and dodecylphosphocholine 1 1 The coordinates of 19 refined structures and the NMR-derived restraints have been deposited in the Brookhaven Protein Data Bank, PDB ID code 1BY6

Biochimica Et Biophysica Acta-molecular and Cell Biology of Lipids, 2000

We have studied the three-dimensional structure of a biologically active peptide of apolipoprotei... more We have studied the three-dimensional structure of a biologically active peptide of apolipoprotein C-II (apoC-II) in the presence of lipid mimetics by CD and NMR spectroscopy. This peptide, corresponding to residues 44–79 of apoC-II, has been shown to reverse the symptoms of genetic apoC-II deficiency in a human subject. A comparison of α-proton secondary shifts and CD spectroscopic data indicates

Research paper thumbnail of Conformation of Human Apolipoprotein CI in a Lipid-Mimetic Environment Determined by CD and NMR Spectroscopy

Biochemistry, 1999

The high-resolution conformation of human apoC-I in complexes with sodium dodecyl sulfate (SDS) i... more The high-resolution conformation of human apoC-I in complexes with sodium dodecyl sulfate (SDS) is presented. As estimated from CD data, apoC-I adopts 54% helical secondary structure when bound to SDS, which is similar to the helical content previously found with phospholipids. The NMR-derived conformation of apoC-I is composed of two amphipathic helices, residues 7-29 and 38-52, separated by a flexible linker. The N-terminal helix contains a mobile hinge involving residues 12-15. The hydrophobic side chains cluster on the nonpolar face of both helices, thus forming two discrete lipid-binding sites in the N-terminal helix and one in the C-terminal helix. As suggested by amide proton resonance line widths and deuterium exchange rates, the N-terminal helix is more flexible and may bind less tightly to the detergent than the C-terminal helix. The different mobility of both helices appears to be related to side-chain composition, rather than length of the amphipathic helix, and may play a role in the function of apoC-I as an activator of lecithin:cholesterol acyltransferase (LCAT). A model is suggested in which the C-terminal helix serves as a lipid anchor while the N-terminal helix may hinge off the lipid surface to make specific contacts with LCAT.

Research paper thumbnail of Conformational studies of the N-terminal lipid-associating domain of human apolipoprotein CI by CD and 1 H NMR spectroscopy

Protein Science, 1997

A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was sy... more A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was synthesized using solid-phase methods and its solution conformation studied by CD and 1H NMR spectroscopy. The CD data indicate that apoC-I(1-38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS-bound apoC-I(1-38) was calculated from 464 NOE-based distance restraints using distance geometry methods. ApoC-I(1-38) adopts a helical structure between residues V4 and K30 and an extended C-terminus from Q31 when associated with SDS. The region K12-G15 undergoes slow conformational exchange as indicated by above-average amide resonance linewidths, large temperature coefficients, and fast exchange (<2 h) of backbone amide protons with deuterium. The mobility of K12-G15 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC-I(1-38) is curved toward its hydrophobic face with bends of 125°, centered at K12/E13, and 150°, centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, I26, and I29, with the amphiphile SDS. Based on our present structural definition of apoC-I(1-38) and the previously obtained structure of the fragment apoC-I(35-53), we propose the secondary structure of intact apolipoprotein C-I.

Research paper thumbnail of The use of sodium dodecyl sulfate to model the apolipoprotein environment. Evidence for peptide–SDS complexes using pulsed-field-gradient NMR spectroscopy

Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1998

Pulsed-field-gradient NMR spectroscopy was used to measure translational diffusion coefficients D... more Pulsed-field-gradient NMR spectroscopy was used to measure translational diffusion coefficients D for a peptide s Ž Ž .. corresponding to a proposed lipid-binding domain of human apolipoprotein C-I, residues 7-24 apoC-I 7-24 . Diffusion Ž . coefficients for apoC-I 7-24 were determined directly by following the decay of the resonance intensity of selected peptide Ž . protons at various concentrations of sodium dodecyl sulfate SDS , a detergent increasingly being used to model the apolipoprotein environment. Previously, diffusion coefficients of peptides in the presence of SDS have been determined indirectly by monitoring the SDS diffusion coefficient. The direct measurement of the diffusion coefficient of the peptide enables one to distinguish whether SDS simply coats the peptide's surface to produce a uniformly charged 'rod' or if the Ž peptide associates with a micelle. Using the direct method, at SDS concentrations above 5 mM which is below the SDS Ž . . Ž . critical micelle concentration 8.1 mM , apoC-I 7-24 exhibited diffusion coefficients consistent with the formation of a large-molecular-weight complex. Based on the ratio of the diffusion coefficients for free-and SDS-associated peptide, the molecular weight of the peptide-SDS complex was much larger than a factor of 1.4, the increase in molecular weight of the Ž . free peptide predicted if apoC-I 7-24 was uniformly surface coated with SDS. q 1998 Elsevier Science B.V.

Research paper thumbnail of Structural Transitions as Determinants of the Action of the Calcium-Dependent Antibiotic Daptomycin

Chemistry & Biology, 2004

Daptomycin is a cyclic anionic lipopeptide antibiotic recently approved for the treatment of comp... more Daptomycin is a cyclic anionic lipopeptide antibiotic recently approved for the treatment of complicated skin infections (Cubicin). Its function is dependent on calcium (as Ca2+). Circular dichroism spectroscopy indicated that daptomycin experienced two structural transitions: a transition upon interaction of daptomycin with Ca2+, and a further transition upon interaction with Ca2+ and the bacterial acidic phospholipid, phosphatidyl glycerol. The Ca2+-dependent insertion of daptomycin into model membranes promoted mild and more pronounced perturbations as assessed by the increase of lipid flip-flop and membrane leakage, respectively. The NMR structure of daptomycin indicated that Ca2+ induced a conformational change in daptomycin that increased its amphipathicity. These results are consistent with the hypothesis that the association of Ca2+ with daptomycin permits it to interact with bacterial membranes with effects that are similar to those of the cationic antimicrobial peptides.

Research paper thumbnail of Structure–activity relationships for the β-hairpin cationic antimicrobial peptide polyphemusin I

Biochimica Et Biophysica Acta-proteins and Proteomics, 2004

The solution structure of polyphemusin I was determined using (1)H-NMR spectroscopy. Polyphemusin... more The solution structure of polyphemusin I was determined using (1)H-NMR spectroscopy. Polyphemusin I was found to be an amphipathic, beta-hairpin connected by a type I&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; beta-turn. The 17 low-energy structures aligned very well over the beta-sheet region while both termini were poorly defined due in part to a hinge-like region centred in the molecule about arginine residues 6 and 16. Conversely, a linear analogue, PM1-S, with all cysteines simultaneously replaced with serine was found to be dynamic in nature, and a lack of medium and long-range NOEs indicated that this molecule displayed no favoured conformation. Circular dichroism (CD) spectroscopy confirmed that in solution, 50% trifluoroethanol (TFE) and in the presence of liposomes, PM1-S remained unstructured. The antimicrobial activity of PM1-S was found to be 4- to 16-fold less than that of polyphemusin I and corresponded with a 4-fold reduction in bacterial membrane depolarization. Both peptides were able to associate with lipid bilayers in a similar fashion; however, PM1-S was completely unable to translocate model membranes while polyphemusin I retained this activity. It was concluded that the disulfide-constrained, beta-sheet structure of polyphemusin I is required for maximum antimicrobial activity. Disruption of this structure results in reduced antimicrobial activity and completely abolishes membrane translocation indicating that the linear PM1-S acts through a different antimicrobial mechanism.

Research paper thumbnail of Role of membranes in the activities of antimicrobial cationic peptides

Fems Microbiology Letters, 2002

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activitie... more Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.

Research paper thumbnail of Structure-Based Design of an Indolicidin Peptide Analogue with Increased Protease Stability

Biochemistry, 2003

Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovin... more Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analogue CP-11, ILKKWPWWPWRRK-NH(2), with improved activity against Gram-negative bacteria had increased positive charge and amphipathicity while maintaining the short length of the parent molecule. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was determined using nuclear magnetic resonance spectroscopy. CP-11 was found to be an amphipathic molecule with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded peptide cycloCP-11, ICLKKWPWWPWRRCK-NH(2), was designed to stabilize the lipid-bound structure and to increase protease resistance. The three-dimensional structure of cycloCP-11 was determined under the same conditions as for the linear peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 associated with phospholipid membranes in a similar manner as indicated by circular dichroism and fluorescence spectra. The minimal inhibitory concentrations of CP-11 and cycloCP-11 for a range of bacteria differed by no more than 2-fold, and they were nonhemolytic at concentrations up to 256 microg/mL. Cyclization was found to greatly increase protease stability. The half-life of cycloCP-11 in the presence of trypsin was increased by 4.5-fold from 4 to 18 min. More importantly, the antibacterial activity of cycloCP-11, but not that of CP-11, in the presence of trypsin was completely retained up to 90 min since the major degradation product was equally active. A structural comparison of CP-11 and cycloCP-11 revealed that the higher trypsin resistance of cycloCP-11 may be due to the more compact packing of lysine and tryptophan side chains. These findings suggest that cyclization may serve as an important strategy in the rational design of antimicrobial peptides.

Research paper thumbnail of Role of membranes in the activities of antimicrobial cationic peptides

Fems Microbiology Letters, 2002

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activitie... more Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.

Research paper thumbnail of Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide

Journal of Biological Chemistry, 2009

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enh... more Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor ␣-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor ␣-induced p62-protein kinase complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancerbinding protein ␤ activity, whereas NF-B activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

Research paper thumbnail of Determination of Membrane Immersion Depth with O 2: A High-Pressure 19F NMR Study

Biophysical Journal, 2001

Oxygen is known to partition with an increasing concentration gradient toward the hydrophobic mem... more Oxygen is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior. At partial pressures (P O2 ) of 100 Atm or more, this concentration gradient is sufficient to induce paramagnetic effects that depend sensitively on membrane immersion depth. This effect is demonstrated for the fluorine nucleus by depthdependent paramagnetic shifts and spin-lattice relaxation rates, using a fluorinated detergent, CF3(CF 2 ) 5 C 2 H 4 -O-maltose (TFOM), reconstituted into a lipid bilayer model membrane system. To interpret the spin-lattice relaxation rates (R 1 P ) in terms of a precise immersion depth, two specifically fluorinated cholesterol species (6-fluorocholesterol and 25-fluorocholesterol), whose membrane immersion depths were independently estimated, were studied by 19 F NMR. The paramagnetic relaxation rates, R 1 P , of the cholesterol species were then used to parameterize a Gaussian profile that directly relates R 1 P to immersion depth z. This same Gaussian curve could then be used to determine the membrane immersion depth of all six fluorinated chain positions of TFOM and of two adjacent residues of specifically fluorinated analogs of the antibacterial peptide indolicidin. The potential of this method for determination of immersion depth and topology of membrane proteins is discussed.

Research paper thumbnail of Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay

Peptides, 2003

Membrane binding and relative penetration of indolicidin analogs were studied using lipid/polydia... more Membrane binding and relative penetration of indolicidin analogs were studied using lipid/polydiacetylene (PDA) chromatic biomimetic membranes. Colorimetric and fluorescence analyses determined that an indolicidin analog with a proline and tryptophan residue substituted with lysines showed more pronounced bilayer surface interactions, while indolicidin and particularly an indolicidin analog in which all prolines were replaced with alanine residues exhibited deeper insertion into the lipid bilayer. The colorimetric data demonstrated that more pronounced blue-red transitions were observed when the chromatic vesicles incorporated lipopolysaccharide (LPS) within the lipid bilayer, indicating that LPS promoted preferred binding and incorporation of the peptides at the lipid/water interface. The fluorescence quenching experiments further confirmed this outcome. The results indicate that the antibacterial activity of indolicidin most likely requires initial binding to the LPS moieties within bacterial membranes, as well as disruption of the bilayer interface. The degree of hemolysis induced by the analogs, on the other hand, correlated to the extent of penetration into the hydrophobic core of the lipid assembly.

Research paper thumbnail of Structure of the Bovine Antimicrobial Peptide Indolicidin Bound to Dodecylphosphocholine and Sodium Dodecyl Sulfate Micelles

Biochemistry, 2000

Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusua... more Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusually rich in tryptophan and proline. Its antimicrobial action involves the bacterial cytoplasmic membrane. Fluorescence and circular dichroism spectra demonstrated the structural similarity of indolicidin in complexes with large unilamellar phospolipid vesicles and with detergent micelles. The structure of indolicidin bound to zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecyl sulfate (SDS) micelles was determined using NMR methods and shown to represent a unique membrane-associated peptide structure. The backbone structure in DPC, well defined between residues 3 and 11, was extended, with two half-turns at residues Lys-5 and Trp-8. The backbone structure in SDS, well defined between residues 5 and 11, was also extended, but lacked the bend in the C-terminal half. Indolicidin in complexes with DPC had a central hydrophobic core composed of proline and tryptophan, which was bracketed by positively charged regions near the peptide termini. The tryptophan side chains, with one exception, folded flat against the peptide backbone, thus giving the molecule a wedge shape. Indolicidin in complexes with SDS had an arrangement of hydrophobic and cationic regions similar to that found in the presence of DPC. The tryptophan side chains were less well defined than for indolicidin in DPC and extended away from the peptide backbone. The preferred location of indolicidin in DPC micelles and lipid bilayers, analyzed using spin-label probes, was at the membrane interface.

Research paper thumbnail of Interaction and Cellular Localization of the Human Host Defense Peptide LL37 with Lung Epithelial Cells

Infection and Immunity, 2005

LL-37 is a human cationic host defense peptide that is an essential component of innate immunity.... more LL-37 is a human cationic host defense peptide that is an essential component of innate immunity. In addition to its modest antimicrobial activity, LL-37 affects the gene expression and behavior of effector cells involved in the innate immune response, although its mode of interaction with eukaryotic cells remains unclear. The interaction of LL-37 with epithelial cells was characterized in tissue culture by using biotinylated LL-37 and confocal microscopy. It was demonstrated that LL-37 was actively taken up into A549 epithelial cells and eventually localized to the perinuclear region. Specific inhibitors were used to demonstrate that the uptake process was not mediated by actin but required elements normally involved in endocytosis and that trafficking to the perinuclear region was dependent on microtubules. By using nonlinear regression analysis, it was revealed that A549 epithelial cells have two receptors for LL-37B, with high and low affinity for LL-37, respectively. These results indicate the mode of interaction of LL-37 with epithelial cells and further our understanding of its role in modulating the innate immune response.

Research paper thumbnail of The role of structure in antibody cross-reactivity between peptides and folded proteins 1 1 Edited by J. A. Wells

Journal of Molecular Biology, 1998

Peptides have the potential for targeting vaccines against pre-speci®ed epitopes on folded protei... more Peptides have the potential for targeting vaccines against pre-speci®ed epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced ®t of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with submicromolar af®nities. The strong preference for a type II b-turn predicted for one peptide was con®rmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.

Research paper thumbnail of The role of structure in antibody cross-reactivity between peptides and folded proteins1

Journal of Molecular Biology, 1998

Peptides have the potential for targeting vaccines against pre-speci®ed epitopes on folded protei... more Peptides have the potential for targeting vaccines against pre-speci®ed epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced ®t of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with submicromolar af®nities. The strong preference for a type II b-turn predicted for one peptide was con®rmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.

Research paper thumbnail of Sequence-specific 1 H NMR resonance assignments and secondary structure of human apolipoprotein CI in the presence of sodium dodecyl sulfate

Biochemistry and Cell Biology-biochimie Et Biologie Cellulaire, 1998

Apolipoprotein (apo) C-I is a 57-residue exchangeable plasma protein distributed mainly in high a... more Apolipoprotein (apo) C-I is a 57-residue exchangeable plasma protein distributed mainly in high and very low density lipoprotein. In this report we present the nuclear magnetic resonance spectra of native apoC-I and synthetic apoC-I, containing selected 15N-labelled amino acids, in the presence of sodium dodecyl sulfate. The proton resonances of apoC-I are assigned and the secondary structure is estimated from the difference of measured alpha-proton chemical shifts to random coil values and the observed NOE interactions. According to these data apoC-I forms two helices, Val-4-Lys-30 and Leu-34-Lys-52, linked by an unstructured region Gln-31-Glu-33. The N-terminal segments of each helix, Val-4-Gly-15 and Leu-34-Met-38, appear to be more flexible than the helical core regions Asn-16-Lys-30 and Arg-39-Lys-52.

Research paper thumbnail of Infrared Spectroscopy of Human Apolipoprotein Fragments in SDS/D 2 O: Relative Lipid-Binding Affinities and a Novel Amide I Assignment

Biochemistry, 1997

Infrared absorption spectra are reported for six apolipoprotein fragments in SDS/D 2 O. Five of t... more Infrared absorption spectra are reported for six apolipoprotein fragments in SDS/D 2 O. Five of the peptides correspond to proposed lipid-binding domains of human apolipoproteins [apoC-I(7-24), apoC-I(35-53), apoA-II(18-30)+, apoA-I(166-185), apoE(267-289)], and the sixth is the de noVo lipid associating peptide LAP-20. The amide I infrared absorption patterns are generally consistent with predominantly helical structures (as determined previously by NMR spectroscopy and distance geometry calculations) and further suggest that apoA-I(166-185) and apoE(267-289) are bound to SDS relatively weakly in comparison to the other four peptides. The latter conclusion is also supported by the temperature dependence of the infrared spectra, as increasing temperature promotes a distinct increase in random coil structure only for apoA-I(166-185) and apoE . In addition to features readily ascribed to helices, the infrared spectra of all the peptides show absorptions in the spectral region 1630-1635 cm -1 that is usually associated with -structure, a motif that is clearly absent from the NMR-derived structures. Parallel difficulties also arose in the analyses of the circular dichroism spectra. We suggest that both the lowfrequency infrared absorptions and the ambiguities in interpreting the CD spectra may be due to unusual structures at the peptide C-termini, involving CdO groups that form hydrogen bonds simultaneously either with two solvent molecules or with donors from the backbone (NH) and the solvent (OH). Analogous absorptions may be a general feature of solvent-exposed helices, which suggests a need for caution in assigning amide I bands below 1640 cm -1 . † Issued as NRCC Publication No. 34783.

Research paper thumbnail of Structural studies of a baboon ( Papio sp. ) plasma protein inhibitor of cholesteryl ester transferase

Protein Science, 2000

A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in... more A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N-terminus of baboon apoC-I. Relative to CETIP, baboon apoC-I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid-phase methods. Using sodium dodecyl sulfate (SDS) to model the lipoprotein environment, the solution structure of CETIP was probed by optical and 1HNMR spectroscopy. Circular dichroism data show that the protein lacks a well-defined structure in water but, upon the addition of SDS, becomes helical (56%). A small blue shift of 8 nm was observed in the intrinsic tryptophan fluorescence of CETIP in the presence of saturating amounts of SDS, suggesting that tryptophan-23 is not buried deeply in the lipid environment. The helical nature of CETIP in the presence of SDS was confirmed by upfield 1Hα secondary shifts and an average solution structure determined by distance geometry/simulated annealing calculations using 476 NOE-based distance restraints. The backbone (N — Cα — Cα = O ) root-mean-square deviation of an ensemble of 17 out of 25 calculated structures superimposed on the average structure was 1.06 ± 0.30 Å using residues V4-P35 and 0.51 ± 0.17 Å using residues A7-S32. Although the side-chain orientations fit the basic description of a class A amphipathic helix, both intramolecular salt bridge formation and “snorkeling” of basic side chains toward the polar face play minor, if any, roles in stabilizing the lipid-bound amphipathic structure. Conformational features of the calculated structures for CETIP are discussed relative to models of CETIP inhibition of cholesteryl ester transferase.

Research paper thumbnail of Conformation of Two Peptides Corresponding to Human Apolipoprotein CI Residues 7-24 and 35-53 in the Presence of Sodium Dodecyl Sulfate by CD and NMR Spectroscopy

Biochemistry, 1995

Peptides corresponding to the proposed lipid-binding domains of human apolipoprotein C-I, residue... more Peptides corresponding to the proposed lipid-binding domains of human apolipoprotein C-I, residues 7-24 (ALDKLKEFGNTLEDKARE) and 35-53 (SAKMREWFSETFQKVKEKL), were studied by CD and two-dimensional 'H NMR spectroscopy. Sodium dodecyl sulfate (SDS) was used to model the lipoprotein environment. Analysis of the CD data shows that both peptides lack well-defined structure in aqueous solution but adopt helical, ordered structures upon the addition of SDS. The helical nature of the peptides in the presence of SDS was confirmed by Ha secondary shifts. A total of 199 (apoC-1(7-24)) and 266 (apoC-I(35-53)) distance restraints were used in distance geometry and simulated annealing calculations to generate average structures for both peptides in aqueous solutions containing SDS. The backbone (N, Ca, C=O) RMSD from the average structure of an ensemble of 20 structures was 0.73 f 0.22 and 0.48 f 0.14 A for apoC-I(7-24) and apoC-1(35-53), respectively. In the presence of SDS, the distance geometry and simulated annealing calculations show that both peptides adopt well-defined amphipathic helices with distinct hydrophobic and hydrophilic faces. The calculated structures are discussed relative to predicted structures. Comparing our CD and NMR results for the apoC-I fragments in SDS with CD results of others obtained in the presence of dimyristoylphosphatidylcholine indicates that SDS may be a better model of the lipoprotein environment.

Research paper thumbnail of Structure of a biologically active fragment of human serum apolipoprotein C-II in the presence of sodium dodecyl sulfate and dodecylphosphocholine 1 1 The coordinates of 19 refined structures and the NMR-derived restraints have been deposited in the Brookhaven Protein Data Bank, PDB ID code 1BY6

Biochimica Et Biophysica Acta-molecular and Cell Biology of Lipids, 2000

We have studied the three-dimensional structure of a biologically active peptide of apolipoprotei... more We have studied the three-dimensional structure of a biologically active peptide of apolipoprotein C-II (apoC-II) in the presence of lipid mimetics by CD and NMR spectroscopy. This peptide, corresponding to residues 44–79 of apoC-II, has been shown to reverse the symptoms of genetic apoC-II deficiency in a human subject. A comparison of α-proton secondary shifts and CD spectroscopic data indicates

Research paper thumbnail of Conformation of Human Apolipoprotein CI in a Lipid-Mimetic Environment Determined by CD and NMR Spectroscopy

Biochemistry, 1999

The high-resolution conformation of human apoC-I in complexes with sodium dodecyl sulfate (SDS) i... more The high-resolution conformation of human apoC-I in complexes with sodium dodecyl sulfate (SDS) is presented. As estimated from CD data, apoC-I adopts 54% helical secondary structure when bound to SDS, which is similar to the helical content previously found with phospholipids. The NMR-derived conformation of apoC-I is composed of two amphipathic helices, residues 7-29 and 38-52, separated by a flexible linker. The N-terminal helix contains a mobile hinge involving residues 12-15. The hydrophobic side chains cluster on the nonpolar face of both helices, thus forming two discrete lipid-binding sites in the N-terminal helix and one in the C-terminal helix. As suggested by amide proton resonance line widths and deuterium exchange rates, the N-terminal helix is more flexible and may bind less tightly to the detergent than the C-terminal helix. The different mobility of both helices appears to be related to side-chain composition, rather than length of the amphipathic helix, and may play a role in the function of apoC-I as an activator of lecithin:cholesterol acyltransferase (LCAT). A model is suggested in which the C-terminal helix serves as a lipid anchor while the N-terminal helix may hinge off the lipid surface to make specific contacts with LCAT.

Research paper thumbnail of Conformational studies of the N-terminal lipid-associating domain of human apolipoprotein CI by CD and 1 H NMR spectroscopy

Protein Science, 1997

A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was sy... more A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was synthesized using solid-phase methods and its solution conformation studied by CD and 1H NMR spectroscopy. The CD data indicate that apoC-I(1-38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS-bound apoC-I(1-38) was calculated from 464 NOE-based distance restraints using distance geometry methods. ApoC-I(1-38) adopts a helical structure between residues V4 and K30 and an extended C-terminus from Q31 when associated with SDS. The region K12-G15 undergoes slow conformational exchange as indicated by above-average amide resonance linewidths, large temperature coefficients, and fast exchange (<2 h) of backbone amide protons with deuterium. The mobility of K12-G15 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC-I(1-38) is curved toward its hydrophobic face with bends of 125°, centered at K12/E13, and 150°, centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, I26, and I29, with the amphiphile SDS. Based on our present structural definition of apoC-I(1-38) and the previously obtained structure of the fragment apoC-I(35-53), we propose the secondary structure of intact apolipoprotein C-I.

Research paper thumbnail of The use of sodium dodecyl sulfate to model the apolipoprotein environment. Evidence for peptide–SDS complexes using pulsed-field-gradient NMR spectroscopy

Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1998

Pulsed-field-gradient NMR spectroscopy was used to measure translational diffusion coefficients D... more Pulsed-field-gradient NMR spectroscopy was used to measure translational diffusion coefficients D for a peptide s Ž Ž .. corresponding to a proposed lipid-binding domain of human apolipoprotein C-I, residues 7-24 apoC-I 7-24 . Diffusion Ž . coefficients for apoC-I 7-24 were determined directly by following the decay of the resonance intensity of selected peptide Ž . protons at various concentrations of sodium dodecyl sulfate SDS , a detergent increasingly being used to model the apolipoprotein environment. Previously, diffusion coefficients of peptides in the presence of SDS have been determined indirectly by monitoring the SDS diffusion coefficient. The direct measurement of the diffusion coefficient of the peptide enables one to distinguish whether SDS simply coats the peptide's surface to produce a uniformly charged 'rod' or if the Ž peptide associates with a micelle. Using the direct method, at SDS concentrations above 5 mM which is below the SDS Ž . . Ž . critical micelle concentration 8.1 mM , apoC-I 7-24 exhibited diffusion coefficients consistent with the formation of a large-molecular-weight complex. Based on the ratio of the diffusion coefficients for free-and SDS-associated peptide, the molecular weight of the peptide-SDS complex was much larger than a factor of 1.4, the increase in molecular weight of the Ž . free peptide predicted if apoC-I 7-24 was uniformly surface coated with SDS. q 1998 Elsevier Science B.V.

Research paper thumbnail of Structural Transitions as Determinants of the Action of the Calcium-Dependent Antibiotic Daptomycin

Chemistry & Biology, 2004

Daptomycin is a cyclic anionic lipopeptide antibiotic recently approved for the treatment of comp... more Daptomycin is a cyclic anionic lipopeptide antibiotic recently approved for the treatment of complicated skin infections (Cubicin). Its function is dependent on calcium (as Ca2+). Circular dichroism spectroscopy indicated that daptomycin experienced two structural transitions: a transition upon interaction of daptomycin with Ca2+, and a further transition upon interaction with Ca2+ and the bacterial acidic phospholipid, phosphatidyl glycerol. The Ca2+-dependent insertion of daptomycin into model membranes promoted mild and more pronounced perturbations as assessed by the increase of lipid flip-flop and membrane leakage, respectively. The NMR structure of daptomycin indicated that Ca2+ induced a conformational change in daptomycin that increased its amphipathicity. These results are consistent with the hypothesis that the association of Ca2+ with daptomycin permits it to interact with bacterial membranes with effects that are similar to those of the cationic antimicrobial peptides.

Research paper thumbnail of Structure–activity relationships for the β-hairpin cationic antimicrobial peptide polyphemusin I

Biochimica Et Biophysica Acta-proteins and Proteomics, 2004

The solution structure of polyphemusin I was determined using (1)H-NMR spectroscopy. Polyphemusin... more The solution structure of polyphemusin I was determined using (1)H-NMR spectroscopy. Polyphemusin I was found to be an amphipathic, beta-hairpin connected by a type I&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; beta-turn. The 17 low-energy structures aligned very well over the beta-sheet region while both termini were poorly defined due in part to a hinge-like region centred in the molecule about arginine residues 6 and 16. Conversely, a linear analogue, PM1-S, with all cysteines simultaneously replaced with serine was found to be dynamic in nature, and a lack of medium and long-range NOEs indicated that this molecule displayed no favoured conformation. Circular dichroism (CD) spectroscopy confirmed that in solution, 50% trifluoroethanol (TFE) and in the presence of liposomes, PM1-S remained unstructured. The antimicrobial activity of PM1-S was found to be 4- to 16-fold less than that of polyphemusin I and corresponded with a 4-fold reduction in bacterial membrane depolarization. Both peptides were able to associate with lipid bilayers in a similar fashion; however, PM1-S was completely unable to translocate model membranes while polyphemusin I retained this activity. It was concluded that the disulfide-constrained, beta-sheet structure of polyphemusin I is required for maximum antimicrobial activity. Disruption of this structure results in reduced antimicrobial activity and completely abolishes membrane translocation indicating that the linear PM1-S acts through a different antimicrobial mechanism.

Research paper thumbnail of Role of membranes in the activities of antimicrobial cationic peptides

Fems Microbiology Letters, 2002

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activitie... more Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.

Research paper thumbnail of Structure-Based Design of an Indolicidin Peptide Analogue with Increased Protease Stability

Biochemistry, 2003

Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovin... more Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analogue CP-11, ILKKWPWWPWRRK-NH(2), with improved activity against Gram-negative bacteria had increased positive charge and amphipathicity while maintaining the short length of the parent molecule. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was determined using nuclear magnetic resonance spectroscopy. CP-11 was found to be an amphipathic molecule with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded peptide cycloCP-11, ICLKKWPWWPWRRCK-NH(2), was designed to stabilize the lipid-bound structure and to increase protease resistance. The three-dimensional structure of cycloCP-11 was determined under the same conditions as for the linear peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 associated with phospholipid membranes in a similar manner as indicated by circular dichroism and fluorescence spectra. The minimal inhibitory concentrations of CP-11 and cycloCP-11 for a range of bacteria differed by no more than 2-fold, and they were nonhemolytic at concentrations up to 256 microg/mL. Cyclization was found to greatly increase protease stability. The half-life of cycloCP-11 in the presence of trypsin was increased by 4.5-fold from 4 to 18 min. More importantly, the antibacterial activity of cycloCP-11, but not that of CP-11, in the presence of trypsin was completely retained up to 90 min since the major degradation product was equally active. A structural comparison of CP-11 and cycloCP-11 revealed that the higher trypsin resistance of cycloCP-11 may be due to the more compact packing of lysine and tryptophan side chains. These findings suggest that cyclization may serve as an important strategy in the rational design of antimicrobial peptides.

Research paper thumbnail of Role of membranes in the activities of antimicrobial cationic peptides

Fems Microbiology Letters, 2002

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activitie... more Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.

Research paper thumbnail of Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide

Journal of Biological Chemistry, 2009

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enh... more Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor ␣-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor ␣-induced p62-protein kinase complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancerbinding protein ␤ activity, whereas NF-B activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

Research paper thumbnail of Determination of Membrane Immersion Depth with O 2: A High-Pressure 19F NMR Study

Biophysical Journal, 2001

Oxygen is known to partition with an increasing concentration gradient toward the hydrophobic mem... more Oxygen is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior. At partial pressures (P O2 ) of 100 Atm or more, this concentration gradient is sufficient to induce paramagnetic effects that depend sensitively on membrane immersion depth. This effect is demonstrated for the fluorine nucleus by depthdependent paramagnetic shifts and spin-lattice relaxation rates, using a fluorinated detergent, CF3(CF 2 ) 5 C 2 H 4 -O-maltose (TFOM), reconstituted into a lipid bilayer model membrane system. To interpret the spin-lattice relaxation rates (R 1 P ) in terms of a precise immersion depth, two specifically fluorinated cholesterol species (6-fluorocholesterol and 25-fluorocholesterol), whose membrane immersion depths were independently estimated, were studied by 19 F NMR. The paramagnetic relaxation rates, R 1 P , of the cholesterol species were then used to parameterize a Gaussian profile that directly relates R 1 P to immersion depth z. This same Gaussian curve could then be used to determine the membrane immersion depth of all six fluorinated chain positions of TFOM and of two adjacent residues of specifically fluorinated analogs of the antibacterial peptide indolicidin. The potential of this method for determination of immersion depth and topology of membrane proteins is discussed.

Research paper thumbnail of Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay

Peptides, 2003

Membrane binding and relative penetration of indolicidin analogs were studied using lipid/polydia... more Membrane binding and relative penetration of indolicidin analogs were studied using lipid/polydiacetylene (PDA) chromatic biomimetic membranes. Colorimetric and fluorescence analyses determined that an indolicidin analog with a proline and tryptophan residue substituted with lysines showed more pronounced bilayer surface interactions, while indolicidin and particularly an indolicidin analog in which all prolines were replaced with alanine residues exhibited deeper insertion into the lipid bilayer. The colorimetric data demonstrated that more pronounced blue-red transitions were observed when the chromatic vesicles incorporated lipopolysaccharide (LPS) within the lipid bilayer, indicating that LPS promoted preferred binding and incorporation of the peptides at the lipid/water interface. The fluorescence quenching experiments further confirmed this outcome. The results indicate that the antibacterial activity of indolicidin most likely requires initial binding to the LPS moieties within bacterial membranes, as well as disruption of the bilayer interface. The degree of hemolysis induced by the analogs, on the other hand, correlated to the extent of penetration into the hydrophobic core of the lipid assembly.

Research paper thumbnail of Structure of the Bovine Antimicrobial Peptide Indolicidin Bound to Dodecylphosphocholine and Sodium Dodecyl Sulfate Micelles

Biochemistry, 2000

Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusua... more Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusually rich in tryptophan and proline. Its antimicrobial action involves the bacterial cytoplasmic membrane. Fluorescence and circular dichroism spectra demonstrated the structural similarity of indolicidin in complexes with large unilamellar phospolipid vesicles and with detergent micelles. The structure of indolicidin bound to zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecyl sulfate (SDS) micelles was determined using NMR methods and shown to represent a unique membrane-associated peptide structure. The backbone structure in DPC, well defined between residues 3 and 11, was extended, with two half-turns at residues Lys-5 and Trp-8. The backbone structure in SDS, well defined between residues 5 and 11, was also extended, but lacked the bend in the C-terminal half. Indolicidin in complexes with DPC had a central hydrophobic core composed of proline and tryptophan, which was bracketed by positively charged regions near the peptide termini. The tryptophan side chains, with one exception, folded flat against the peptide backbone, thus giving the molecule a wedge shape. Indolicidin in complexes with SDS had an arrangement of hydrophobic and cationic regions similar to that found in the presence of DPC. The tryptophan side chains were less well defined than for indolicidin in DPC and extended away from the peptide backbone. The preferred location of indolicidin in DPC micelles and lipid bilayers, analyzed using spin-label probes, was at the membrane interface.

Research paper thumbnail of Interaction and Cellular Localization of the Human Host Defense Peptide LL37 with Lung Epithelial Cells

Infection and Immunity, 2005

LL-37 is a human cationic host defense peptide that is an essential component of innate immunity.... more LL-37 is a human cationic host defense peptide that is an essential component of innate immunity. In addition to its modest antimicrobial activity, LL-37 affects the gene expression and behavior of effector cells involved in the innate immune response, although its mode of interaction with eukaryotic cells remains unclear. The interaction of LL-37 with epithelial cells was characterized in tissue culture by using biotinylated LL-37 and confocal microscopy. It was demonstrated that LL-37 was actively taken up into A549 epithelial cells and eventually localized to the perinuclear region. Specific inhibitors were used to demonstrate that the uptake process was not mediated by actin but required elements normally involved in endocytosis and that trafficking to the perinuclear region was dependent on microtubules. By using nonlinear regression analysis, it was revealed that A549 epithelial cells have two receptors for LL-37B, with high and low affinity for LL-37, respectively. These results indicate the mode of interaction of LL-37 with epithelial cells and further our understanding of its role in modulating the innate immune response.