Self-assembly of peptide nanocapsules by a solvent concentration gradient.

Biological systems can create materials with intricate structures and specialized functions. In comparison, precise control of structures in human-made materials has been challenging. Here we report on insect cuticle peptides that spontaneously form nanocapsules through a single-step solvent exchange process, where the concentration gradient resulting from the mixing of water and acetone drives the localization and self-assembly of the peptides into hollow nanocapsules. The underlying driving force is found to be the intrinsic affinity of the peptides for a particular solvent concentration, while the diffusion of water and acetone creates a gradient interface that triggers peptide localization and self-assembly. This gradient-mediated self-assembly offers a transformative pathway towards simple generation of drug delivery systems based on peptide nanocapsules.

Compliant Clients: Catechols Exhibit Enhanced Solubility and Stability in Diverse Complex Coacervates.

Polyelectrolyte coacervates, with their greater-than-water density, low interfacial energy, shear thinning viscosity, and ability to undergo structural arrest, mediate the formation of diverse load-bearing macromolecular materials in living organisms as well as in industrial material fabrication. Coacervates, however, have other useful attributes that are challenging to study given the metastability of coacervate colloidal droplets and a lack of suitable analytical methods. We adopt solution electrochemistry and nuclear magnetic resonance measurements to obtain remarkable insights about coacervates as solvent media for low-molecular-weight catechols. When catechols are added to dispersions of coacervated polyelectrolytes, there are two significant consequences: (1) catechols preferentially partition up to 260-fold into the coacervate phase, and (2) coacervates stabilize catechol redox potentials by up to +200 mV relative to the equilibrium solution. The results suggest that the relationship between phase-separated polyelectrolytes and their client molecules is distinct from that existing in aqueous solution and has the potential for insulating many redox-unstable chemicals.

Release of frustration drives corneal amyloid disaggregation by brain chaperone.

TGFBI-related corneal dystrophy (CD) is characterized by the accumulation of insoluble protein deposits in the corneal tissues, eventually leading to progressive corneal opacity. Here we show that ATP-independent amyloid-ß chaperone L-PGDS can effectively disaggregate corneal amyloids in surgically excised human cornea of TGFBI-CD patients and release trapped amyloid hallmark proteins. Since the mechanism of amyloid disassembly by ATP-independent chaperones is unknown, we reconstructed atomic models of the amyloids self-assembled from TGFBIp-derived peptides and their complex with L-PGDS using cryo-EM and NMR. We show that L-PGDS specifically recognizes structurally frustrated regions in the amyloids and releases those frustrations. The released free energy increases the chaperone's binding affinity to amyloids, resulting in local restructuring and breakage of amyloids to protofibrils. Our mechanistic model provides insights into the alternative source of energy utilized by ATP-independent disaggregases and highlights the possibility of using these chaperones as treatment strategies for different types of amyloid-related diseases.

Liquid-Liquid Phase Separation of the Green Mussel Adhesive Protein Pvfp-5 is Regulated by the Post-Translated Dopa Amino Acid.

The underwater adhesive prowess of aquatic mussels has been largely attributed to the abundant post-translationally modified amino acid l-3,4-dihydroxyphenylalanine (Dopa) in mussel foot proteins (MFPs) that make up their adhesive threads. More recently, it has been suggested that during thread fabrication, MFPs form intermediate fluidic phases such as liquid crystals or coacervates regulated by a liquid-liquid phase separation (LLPS) process. Here, it is shown that Dopa plays another central role during mussel fiber formation, by enabling LLPS of Pvfp-5ß, a main MFP of the green mussel Perna viridis. Using residue-specific substitution of Tyrosine (Tyr) for Dopa during recombinant expression, Dopa-substituted Pvfp-5ß is shown to exhibit LLPS under seawater-like conditions, whereas the Tyr-only version forms insoluble aggregates. Combining quantum chemistry calculations and solution NMR, a transient H-bonding network requiring the two hydroxyl groups of Dopa is found to be critical to enable LLPS in Dopa-mutated Pvfp-5ß. Overall, the study suggests that Dopa plays an important role in regulating LLPS of MFPs, which may be critical to concentrate the adhesive proteins at the plaque/substrate interface and therefore produce a more robust adhesive. The findings also provide molecular-level lessons to guide biomanufacturing of protein-based materials such as bioadhesives and load-bearing fibers.

Structure of a consensus chitin-binding domain revealed by solution NMR.

Chitin-binding proteins (CBPs) are a versatile group of proteins found in almost every organism on earth. CBPs are involved in enzymatic carbohydrate degradation and also serve as templating scaffolds in the exoskeleton of crustaceans and insects. One specific chitin-binding motif found across a wide range of arthropods' exoskeletons is the "extended Rebers and Riddiford" consensus (R&R), whose mechanism of chitin binding remains unclear. Here, we report the 3D structure and molecular level interactions of a chitin-binding domain (CBD-γ) located in a CBP from the beak of the jumbo squid Dosidicus gigas. This CBP is one of four chitin-binding proteins identified in the beak mouthpart of D. gigas and is believed to interact with chitin to form a scaffold network that is infiltrated with a second set of structural proteins during beak maturation. We used solution state NMR spectroscopy to elucidate the molecular interactions between CBD-γ and the soluble chitin derivative pentaacetyl-chitopentaose (PCP), and find that folding of CBD-γ is triggered upon its interaction with PCP. To our knowledge, this is the first experimental 3D structure of a CBP containing the R&R consensus motif, which can be used as a template to understand in more details the role of the R&R motif found in a wide range of CBP-chitin complexes. The present structure also provides molecular information for biomimetic synthesis of graded biomaterials using aqueous-based chemistry and biopolymers.

A Short Peptide Hydrogel with High Stiffness Induced by 310-Helices to ß-Sheet Transition in Water.

Biological gels generally require polymeric chains that produce long-lived physical entanglements. Low molecular weight colloids offer an alternative to macromolecular gels, but often require ad-hoc synthetic procedures. Here, a short biomimetic peptide composed of eight amino acid residues derived from squid sucker ring teeth proteins is demonstrated to form hydrogel in water without any cross-linking agent or chemical modification and exhibits a stiffness on par with the stiffest peptide hydrogels. Combining solution and solid-state NMR, circular dichroism, infrared spectroscopy, and X-ray scattering, the peptide is shown to form a supramolecular, semiflexible gel assembled from unusual right-handed 310-helices stabilized in solution by π-π stacking. During gelation, the 310-helices undergo conformational transition into antiparallel ß-sheets with formation of new interpeptide hydrophobic interactions, and molecular dynamic simulations corroborate stabilization by cross ß-sheet oligomerization. The current study broadens the range of secondary structures available to create supramolecular hydrogels, and introduces 310-helices as transient building blocks for gelation via a 310-to-ß-sheet conformational transition.

Three-dimensional structure of Megabalanus rosa Cement Protein 20 revealed by multi-dimensional NMR and molecular dynamics simulations.

Barnacles employ a protein-based cement to firmly attach to immersed substrates. The cement proteins (CPs) have previously been identified and sequenced. However, the molecular mechanisms of adhesion are not well understood, in particular, because the three-dimensional molecular structure of CPs remained unknown to date. Here, we conducted multi-dimensional nuclear magnetic resonance (NMR) studies and molecular dynamics (MD) simulations of recombinant Megabalanus rosa Cement Protein 20 (rMrCP20). Our NMR results show that rMrCP20 contains three main folded domain regions intervened by two dynamic loops, resulting in multiple protein conformations that exist in equilibrium. We found that 12 out of 32 Cys in the sequence engage in disulfide bonds that stabilize the ß-sheet domains owing to their placement at the extremities of ß-strands. Another feature unveiled by NMR is the location of basic residues in turn regions that are exposed to the solvent, playing an important role for intermolecular contact with negatively charged surfaces. MD simulations highlight a highly stable and conserved ß-motif (ß7-ß8), which may function as nuclei for amyloid-like nanofibrils previously observed in the cured adhesive cement. To the best of our knowledge, this is the first report describing the tertiary structure of an extracellular biological adhesive protein at the molecular level. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Supramolecular propensity of suckerin proteins is driven by ß-sheets and aromatic interactions as revealed by solution NMR.

Suckerin proteins are a family of block co-polymer-like structural proteins that self-assemble into robust supramolecular structures - the sucker ring teeth (SRT) - which are located on the arms and tentacles of cephalopods and used to firmly capture preys. Suckerins are promising biomimetic protein-based biopolymers, but the supramolecular interactions stabilizing SRT remain unknown. Here, we report multi-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy structural studies of an engineered suckerin protein composed of two main sequence modules. The protein adopts a dynamic structure with regions in both module 1 (M1: residues A42-A52) and module 2 (M2: residues G30-Y37 and G58-Y65) folding into anti-parallel ß-sheets and displaying ß-strand propensity, respectively. The obtained structure highlights that aromatic residues present in glycine (Gly)-rich M2 modules are involved in π-π stacking interactions, leading to the stabilization of the structural core. In addition, hydrogen/deuterium (H/D) exchange studies demonstrate a high protection of residues involved in intra-molecular ß-sheets. Gaining a better understanding of the molecular structure of suckerin provides key molecular lessons that may be mimicked in the de novo design of peptide- and protein-based biomaterials with applications in medicine, tissue engineering and nanotechnology.

Fast and Green Synthesis of an Oligo-Hydrocaffeic Acid-Based Adhesive.

A green, mussel-inspired bioadhesive based on oligomerization of hydrocaffeic acid was synthesized in water by an ultrafast one-step reaction in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as an activating agent. The resulting oligomers exhibited strong wet adhesion when applied to different substrates including glass, stainless steel, and aluminum. Compared to most commercial adhesives, this bioinspired adhesive is produced via a sustainable and green process, i.e., aqueous-based synthesis, one-step reaction, and in the absence of any purification step to obtain the final functional adhesive.

Supramolecular ß-Sheets Stabilized Protein Nanocarriers for Drug Delivery and Gene Transfection.

Suckerin proteins, recently discovered in the sucker ring teeth of squids, represent a family of promising structural biomacromolecules that can form supramolecular networks stabilized by nanoconfined ß-sheets. Exploiting this feature as well as their specific amino acid composition, we demonstrate that artificial suckerin-19 (S-19) can be engineered into nanocarriers for efficient drug delivery and gene transfection in vitro and in vivo. First, we demonstrate that S-19 self-assembles into ß-sheet stabilized nanoparticles with controlled particle sizes of 100-200 nm that are able to encapsulate hydrophobic drugs for pH-dependent release in vitro, and that can effectively inhibit tumor growth in vivo. We also show that S-19 can complex and stabilize plasmid DNA, with the complexes stabilized by hydrophobic interactions of the ß-sheet domains as opposed to electrostatic interactions commonly achieved with cationic polymers, thus lowering cytotoxicity. The elevated Histidine content of S-19 appears critical to trigger endosomal escape by the proton sponge effect, thereby ensuring efficient gene transfection both in vitro and in vivo. Our study demonstrates that S-19 represents a promising functional protein nanocarrier that could be used for various drug and gene delivery applications.

'Lollipop'-shaped helical structure of a hybrid antimicrobial peptide of temporin B-lipopolysaccharide binding motif and mapping cationic residues in antibacterial activity.

BACKGROUND: Temporins are attractive templates for the development of antibiotics. However, many temporins are inactive against Gram-negative bacteria. Previously, we demonstrated conjugation of a lipopolysaccharide binding motif peptide to temporins yielded hybrid non-haemolytic AMPs that killed several Gram-negative bacteria. METHODS: We carried out a systematic Ala replacement of individual cationic and polar amino acid residues of LG21, a hybrid AMP consisted of temporin B (TB) and LPS binding motif. These Ala containing analogs of LG21 were examined for antibacterial activity, cell membrane permeabilization and liposome leakage assays using optical spectroscopic methods. Atomic resolution structure of LG21 was determined in zwitterionic dodecyl phosphocholine (DPC) micelles by NMR spectroscopy. RESULTS: Cationic residues in the LPS binding motif of LG21 were critical for bactericidal and membrane permeabilization. Detergent bound structure of LG21 revealed helical conformation containing extensive sidechain/sidechain packing including cation/π interactions in the LPS binding motif. The helical structure of LG21 resembled a 'lollipop' like shape that was sustained by a compacted bulky aromatic/cationic head with a comparatively thinner 'stick' at the N-terminal region. The 'head' of the structure could be localized into micelle-water interfacial region whereas the 'stick' region may be inserted into the hydrophobic core of micelle. CONCLUSIONS: The LPS binding motif of LG21 played dominant roles in broad spectrum activity and the 3-D structure provided plausible mechanistic insights for permeabilization of bacterial membrane. GENERAL SIGNIFICANCE: Hybrid AMPs containing LPS binding motif could be useful for the structure based development of broad spectrum antibiotics.

Salt-resistant short antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising leads for the development of antibiotics against drug resistant bacterial pathogens. However, in vivo applications of AMPs remain obscure due to salt and serum mediated inactivation. The high cost of chemical synthesis of AMPs also impedes potential clinical application. Consequently, short AMPs resistant toward salt and serum inactivation are desirable for the development of peptide antibiotics. In this work, we designed a 12-residue amphipathic helical peptide RR12 (R-R-L-I-R-L-I-L-R-L-L-R-amide) and two Trp containing analogs of RR12 namely RR12Wpolar (R-R-L-I-W-L-I-L-R-L-L-R-amide), and RR12Whydro (R-R-L-I-R-L-W-L-R-L-L-R-amide). Designed peptides demonstrated potent antibacterial activity; MIC ranging from 2 to 8 µM, in the presence of sodium chloride (150 mM and 300 mM). Antibacterial activity of these peptides was also detected in the presence of human serum. Designed peptides, in particular RR12 and RR12Whydro, were only poorly hemolytic. As a mode of action; these peptides demonstrated efficient permeabilization of bacterial cell membrane and lysis of cell structure. We further investigated interactions of the designed peptides with lipopolysaccharide (LPS), the major component of the outer membrane permeability barrier of Gram-negative bacteria. Designed peptides adopted helical conformations in complex with LPS. Binding of peptides with LPS has yielded dissociation the aggregated structures of LPS. Collectively, these designed peptides hold ability to be developed for salt-resistant antimicrobial compounds. Most importantly, current work provides insights for designing salt-resistant antimicrobial peptides. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 345-356, 2016.

Cysteine deleted protegrin-1 (CDP-1): anti-bacterial activity, outer-membrane disruption and selectivity.

BACKGROUND: Protegin-1 (PG-1: RGGRLCYCRRRFCVCVGR-amide) assumes a rigid ß-hairpin like structure that is stabilized by two disulfide bridges between Cys6-Cys15 and Cys8-Cys13. Previous studies, employing linear analogs of PG-1, with Cys to Ala mutations or modified Cys, have demonstrated that the disulfide bridges are critical for the broad spectrum and salt resistant antimicrobial activity of PG-1. METHODS: In order to understand structural and functional roles of disulfide bonds in protegrins, we have synthesized a Cys deleted variant of PG-1 or CDP-1, RGGRLYRRRFVVGR-amide, and two of its analogs, RR11, RLYRRRFVVGR-amide, and LR10, LYRRRFVVGR-amide, containing deletion of residues at the N-terminus. These peptides have been characterized for bactericidal activity and mode of action in lipopolysaccharide (LPS) using optical spectroscopy, ITC and NMR. RESULTS: Antibacterial activity, against Gram-negative and Gram-positive strains, of the three peptides follows the order: CDP-1>RR11>LR10. LR10 displays only limited activity toward Gram-negative strains. CDP-1 demonstrates efficient membrane permeabilization and high-affinity interactions with LPS. CDP-1 and RR11 both assume ß-hairpin like compact structures in complex with LPS, whereas LR10 adopts an extended conformation in LPS. In zwitterionic DPC micelles CDP-1 and the truncated analog peptides do not adopt folded conformations. MAJOR CONCLUSIONS: Despite the absence of stabilizing disulfide bridges CDP-1 shows broad-spectrum antibacterial activity and assumes ß-hairpin like structure in complex with LPS. The ß-hairpin structure may be essential for outer membrane permeabilization and cell killing.

ß-Boomerang Antimicrobial and Antiendotoxic Peptides: Lipidation and Disulfide Bond Effects on Activity and Structure.

Drug-resistant Gram-negative bacterial pathogens and endotoxin- or lipopolysaccharide (LPS)-mediated inflammations are among some of the most  prominent health issues globally. Antimicrobial peptides (AMPs) are eminent molecules that can kill drug-resistant strains and neutralize LPS toxicity. LPS, the outer layer of the outer membrane of Gram-negative bacteria safeguards cell integrity against hydrophobic compounds, including antibiotics and AMPs. Apart from maintaining structural integrity, LPS, when released into the blood stream, also induces inflammatory pathways leading to septic shock. In previous works, we have reported the de novo design of a set of 12-amino acid long cationic/hydrophobic peptides for LPS binding and activity. These peptides adopt ß-boomerang like conformations in complex with LPS. Structure-activity studies demonstrated some critical features of the ß-boomerang scaffold that may be utilized for the further development of potent analogs. In this work, ß-boomerang lipopeptides were designed and structure-activity correlation studies were carried out. These lipopeptides were homo-dimerized through a disulfide bridge to stabilize conformations and for improved activity. The designed peptides exhibited potent antibacterial activity and efficiently neutralized LPS toxicity under in vitro assays. NMR structure of C4YI13C in aqueous solution demonstrated the conserved folding of the lipopeptide with a boomerang aromatic lock stabilized with disulfide bond at the C-terminus and acylation at the N-terminus. These lipo-peptides displaying bacterial sterilization and low hemolytic activity may be useful for future applications as antimicrobial and antiendotoxin molecules.

Resurrecting inactive antimicrobial peptides from the lipopolysaccharide trap.

Host defense antimicrobial peptides (AMPs) are a promising source of antibiotics for the treatment of multiple-drug-resistant pathogens. Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, functions as a permeability barrier against a variety of molecules, including AMPs. Further, LPS or endotoxin is the causative agent of sepsis killing 100,000 people per year in the United States alone. LPS can restrict the activity of AMPs inducing aggregations at the outer membrane, as observed for frog AMPs, temporins, and also in model AMPs. Aggregated AMPs, "trapped" by the outer membrane, are unable to traverse the cell wall, causing their inactivation. In this work, we show that these inactive AMPs can overcome LPS-induced aggregations while conjugated with a short LPS binding ß-boomerang peptide motif and become highly bactericidal. The generated hybrid peptides exhibit activity against Gram-negative and Gram-positive bacteria in high-salt conditions and detoxify endotoxin. Structural and biophysical studies establish the mechanism of action of these peptides in LPS outer membrane. Most importantly, this study provides a new concept for the development of a potent broad-spectrum antibiotic with efficient outer membrane disruption as the mode of action.

Design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt-resistance, and biocompatibility.

Antimicrobial peptides (AMPs) kill microbes by non-specific membrane permeabilization, making them ideal templates for designing novel peptide-based antibiotics that can combat multi-drug resistant pathogens. For maximum efficacy in vivo and in vitro, AMPs must be biocompatible, salt-tolerant and possess broad-spectrum antimicrobial activity. These attributes can be obtained by rational design of peptides guided by good understanding of peptide structure-function. Toward this end, this study investigates the influence of charge and hydrophobicity on the activity of tryptophan and arginine rich decamer peptides engineered from a salt resistant human ß-defensin-28 variant. Mechanistic investigations of the decamers with detergents mimicking the composition of bacterial and mammalian membrane, reveal a correlation between improved antibacterial activity and the increase in tryptophan and positive residue content, while keeping hemolysis low. The potent antimicrobial activity and high cell membrane selective behavior of the two most active decamers, D5 and D6, are attributed to an optimum peptide charge to hydrophobic ratio bestowed by systematic arginine and tryptophan substitution. D5 and D6 show surface localization behavior with binding constants of 1.86 × 10(8) and 2.6 × 10(8) M(-1) , respectively, as determined by isothermal calorimetry measurements. NMR derived structures of D5 and D6 in SDS detergent micelles revealed proximity of Trp and Arg residues in an extended structural scaffold. Such potential cation-π interactions may be critical in cell permeabilization of the AMPs. The fundamental characterization of the engineered decamers provided in this study improves the understanding of structure-activity relationship of short arginine tryptophan rich AMPs, which will pave the way for future de novo design of potent AMPs for therapeutic and biomedical applications.

NMR structure of temporin-1 ta in lipopolysaccharide micelles: mechanistic insight into inactivation by outer membrane.

BACKGROUND: Antimicrobial peptides (AMPs) play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS). The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane. PRINCIPAL FINDINGS: Using NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide) in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD) NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles. SIGNIFICANCE: We propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a broader spectrum of activity.

NMR structure, localization, and vesicle fusion of Chikungunya virus fusion peptide.

The virus-host cell fusion process is mediated by a membrane anchored viral fusion protein that inserts its hydrophobic fusion peptide into the plasma membrane of the host cell, initiating the fusion reaction. Therefore, fusion peptides are an important functional constituent of the fusion proteins of enveloped viruses. In this work, we characterize the fusion peptide or VT18 (V(84)YPFMWGGAYCFCDAENT(101)) of Chikungunya virus (CHIKV) using NMR and fluorescence spectroscopy in zwitterionic lipid environments. Our results demonstrate that the VT18 peptide is able to induce liposome fusions in a pH independent manner and interacts with the zwitterionic lipid vesicles. The NMR derived three-dimensional structure of VT18, in solution of dodecylphosphocholine (DPC) micelles, is typified by extended or ß-type conformations for most of the residues, whereby residues M88-W89-G90-G91 adopt a type I ß-turn conformation. Strikingly, the aromatic side chains of residues Y85, F87, Y93, and F95 in the VT18 structure are found to be well-packed forming an aromatic core. In particular, residue F87 is situated at the center of the aromatic core establishing a close proximity with other aromatic side chains. Further, the aromatic core residues are also involved in packing interactions with the side chains of residues M88, C94. Paramagnetic relaxation enhancement NMR, using spin labeled doxyl lipids, indicated that the aromatic core residues of VT18 are well inserted into the micelles, whereas the polar residues at the C-terminus may be surface localized. The atomic resolution structure and lipid interactions of CHIKV fusion peptide presented here will aid to uncover the fusion mechanism by the type II viral fusion proteins.

Lipopolysaccharide neutralizing peptide-porphyrin conjugates for effective photoinactivation and intracellular imaging of gram-negative bacteria strains.

A simple and specific strategy based on the bioconjugation of a photosensitizer protophophyrin IX (PpIX) with a lipopolysaccharide (LPS) binding antimicrobial peptide YI13WF (YVLWKRKRKFCFI-Amide) has been developed for the effective fluorescent imaging and photodynamic inactivation of Gram-negative bacterial strains. The intracellular fluorescent imaging and photodynamic antimicrobial chemotherapy (PACT) studies supported our hypothesis that the PpIX-YI13WF conjugates could serve as efficient probes to image the bacterial strains and meanwhile indicated the potent activities against Gram-negative bacterial pathogens especially for those with antibiotics resistance when exposed to the white light irradiation. Compared to the monomeric PpIX-YI13WF conjugate, the dimeric conjugate indicated the stronger fluorescent imaging signals and higher photoinactivation toward the Gram-negative bacterial pathogens throughout the whole concentration range. In addition, the photodynamic bacterial inactivation also demonstrated more potent activity than the minimum inhibitory concentration (MIC) values of dimeric PpIX-YI13WF conjugate itself observed for E. coli DH5a (~4 times), S. enterica (~8 times), and other Gram-negative strains including antibiotic-resistant E. coli BL21 (~8 times) and K. pneumoniae (~16 times). Moreover, both fluorescent imaging and photoinactivation measurements also demonstrated that the dimeric PpIX-YI13WF conjugate could selectively recognize bacterial strains over mammalian cells and generate less photo damage to mammalian cells. We believed that the enhanced fluorescence and bacterial inactivation were probably attributed to the higher binding affinity between dimeric photosensitizer peptide conjugate and LPS components on the surface of bacterial strains, which were the results of efficient multivalent interactions.

Structure, activity and interactions of the cysteine deleted analog of tachyplesin-1 with lipopolysaccharide micelle: Mechanistic insights into outer-membrane permeabilization and endotoxin neutralization.

Tachyplesin-1, a disulfide stabilized beta-hairpin antimicrobial peptide, can be found at the hemocytes of horse shoe crab Tachypleus tridentatus. A cysteine deleted linear analog of tachyplesin-1 or CDT (KWFRVYRGIYRRR-NH2) contains a broad spectrum of bactericidal activity with a reduced hemolytic property. The bactericidal activity of CDT stems from selective interactions with the negatively charged lipids including LPS. In this work, CDT-LPS interactions were investigated using NMR spectroscopy, optical spectroscopy and functional assays. We found that CDT neutralized LPS and disrupted permeability barrier of the outer membrane. Zeta potential and ITC studies demonstrated charge compensation and hydrophobic interactions of CDT with the LPS-outer membrane, respectively. Secondary structure of the peptide was probed by CD and FT-IR experiments indicating beta-strands and/or beta-turn conformations in the LPS micelle. An ensemble of structures, determined in LPS micelle by NMR, revealed a beta-hairpin like topology of the CDT peptide that was typified by an extended cationic surface and a relatively shorter segment of hydrophobic region. Interestingly, at the non-polar face, residue R11 was found to be in a close proximity to the indole ring of W2, suggesting a cation-n type interactions. Further, saturation transfer difference (STD) NMR studies established intimate contacts among the aromatic and cationic residues of CDT with the LPS micelle. Fluorescence and dynamic light scattering experiments demonstrated that CDT imparted structural destabilization to the aggregated states of LPS. Collectively, atomic resolution structure and interactions of CDT with the outer membrane-LPS could be exploited for developing potent broad spectrum antimicrobial and anti-sepsis agents.