Diffusion NMR and Rheology of a Model Polymer in Bacterial Cell Lysate Crowders.

The intracellular milieu is crowded and heterogeneous, and this can have profound consequences for biomolecule motions and biochemical kinetics. Macromolecular crowding has been traditionally studied in artificial crowders like Ficoll and dextran or globular proteins such as bovine serum albumin. It is, however, not clear if the effects of artificial crowders on such phenomena are the same as the crowding that is experienced in a heterogeneous biological environment. Bacterial cells, for example, are composed of heterogeneous biomolecules with different sizes, shapes, and charges. Using crowders composed of one of three different pretreatments of bacterial cell lysate (unmanipulated, ultracentrifuged, and anion exchanged), we examine the effects of crowding on the diffusivity of a model polymer. We measure the translational diffusivity, via diffusion NMR, of the test polymer polyethylene glycol (PEG) in these bacterial cell lysates. We show that the small (Rg ∼ 5 nm) test polymer shows a modest decrease in self-diffusivity with increasing crowder concentration for all lysate treatments. The corresponding self-diffusivity decrease in the artificial Ficoll crowder is much more pronounced. Moreover, a comparison of the rheological response of biological and artificial crowders shows that while the artificial crowder Ficoll exhibits a Newtonian response even at high concentrations, the bacterial cell lysate is markedly non-Newtonian; it behaves like a shear-thinning fluid with a yield stress. While at any concentration the rheological properties are sensitive to both lysate pretreatment and batch-to-batch variations, the PEG diffusivity is nearly unaffected by the type of lysate pretreatment.

A pathogenic deletion in Forkhead Box L1 (FOXL1) identifies the first otosclerosis (OTSC) gene.

Otosclerosis is a bone disorder of the otic capsule and common form of late-onset hearing impairment. Considered a complex disease, little is known about its pathogenesis. Over the past 20 years, ten autosomal dominant loci (OTSC1-10) have been mapped but no genes identified. Herein, we map a new OTSC locus to a 9.96 Mb region within the FOX gene cluster on 16q24.1 and identify a 15 bp coding deletion in Forkhead Box L1 co-segregating with otosclerosis in a Caucasian family. Pre-operative phenotype ranges from moderate to severe hearing loss to profound sensorineural loss requiring a cochlear implant. Mutant FOXL1 is both transcribed and translated and correctly locates to the cell nucleus. However, the deletion of 5 residues in the C-terminus of mutant FOXL1 causes a complete loss of transcriptional activity due to loss of secondary (alpha helix) structure. FOXL1 (rs764026385) was identified in a second unrelated case on a shared background. We conclude that FOXL1 (rs764026385) is pathogenic and causes autosomal dominant otosclerosis and propose a key inhibitory role for wildtype Foxl1 in bone remodelling in the otic capsule. New insights into the molecular pathology of otosclerosis from this study provide molecular targets for non-invasive therapeutic interventions.

Antimicrobial Peptide Mechanisms Studied by Whole-Cell Deuterium NMR.

Much of the work probing antimicrobial peptide (AMP) mechanisms has focussed on how these molecules permeabilize lipid bilayers. However, AMPs must also traverse a variety of non-lipid cell envelope components before they reach the lipid bilayer. Additionally, there is a growing list of AMPs with non-lipid targets inside the cell. It is thus useful to extend the biophysical methods that have been traditionally applied to study AMP mechanisms in liposomes to the full bacteria, where the lipids are present along with the full complexity of the rest of the bacterium. This review focusses on what can be learned about AMP mechanisms from solid-state NMR of AMP-treated intact bacteria. It also touches on flow cytometry as a complementary method for measuring permeabilization of bacterial lipid membranes in whole bacteria.

Role of Charge in Lipid Vesicle Binding and Vesicle Surface Saturation by Gaduscidin-1 and Gaduscidin-2.

The histidine-rich antimicrobial peptides Gad-1 and Gad-2, from paralogous genes in cod, provide an opportunity to examine the effect of charge and nonelectrostatic factors on peptide-vesicle interaction and on peptide antimicrobial activity. In this study, the dependence of vesicle ζ-potential on peptide concentration has been used to examine the binding of these peptides to model vesicle surfaces at pH = 5.0, for which the charges of Gad-1 and Gad-2 are +8 and +5, respectively, and at pH = 7.0, where their charges are +3 and +1, respectively. Interpreting the observed ζ-potential behaviors as examples of Langmuir adsorption isotherms, it is possible to infer the equilibrium constant for peptide-vesicle binding, the fraction of the peptide bound at low peptide concentration, and the maximum peptide-to-lipid ratio when the vesicle surface is saturated at high peptide concentration. For both peptides, higher peptide charge is found to be correlated with a lower fraction of the peptide being bound to vesicle surfaces at low peptide concentration and with a smaller maximum bound peptide-to-lipid ratio at high peptide concentration. The equilibrium binding constant, on the other hand, is more strongly correlated with the peptide sequence than with the charge. Gad-1, which has been shown to be more biologically active than Gad-2, displayed a significantly higher equilibrium binding constant. These observations suggest that while the maximum peptide density on the vesicle surface is limited by electrostatic interactions, the free energy of peptide binding, like the observed antimicrobial activities of the Gad peptides, is also sensitive to other peptide factors which might, for example, influence hydrophobic interactions.

All-Atom Molecular Dynamics Simulations of Dimeric Lung Surfactant Protein B in Lipid Multilayers.

Although lung surfactant protein B (SP-B) is an essential protein that plays a crucial role in breathing, the details of its structure and mechanism are not well understood. SP-B forms covalent homodimers, and in this work we use all-atom molecular dynamics simulations to study dimeric SP-B's structure and its behavior in promoting lipid structural transitions. Four initial system configurations were constructed based on current knowledge of SP-B's structure and mechanism, and the protein maintained a helicity consistent with experiment in all systems. Several SP-B-induced lipid reorganization behaviors were observed, and regions of the protein particularly important for these activities included SP-B's "central loop" and "hinge" regions. SP-B dimers with one subunit initially positioned in each of two adjacent bilayers appeared to promote close contact between two bilayers. When both subunits were initially positioned in the same bilayer, SP-B induced the formation of a defect in the bilayer, with water penetrating into the centre of the bilayer. Similarly, dimeric SP-B showed a propensity to interact with preformed interpores in the bilayer. SP-B dimers also promoted bilayer thinning and creasing. This work fleshes out the atomistic details of the dimeric SP-B structures and SP-B/lipid interactions that underlie SP-B's essential functions.

Effects of Amphipathic Polypeptides on Membrane Organization Inferred from Studies Using Bicellar Lipid Mixtures.

SP-B63-78, a lung surfactant protein fragment, and magainin 2, an antimicrobial peptide, are amphipathic peptides with the same overall charge but different biological functions. Deuterium nuclear magnetic resonance has been used to compare the interactions of these peptides with dispersions of 1,2-dimyristoyl- sn-glycero-3-phophocholine (DMPC)/1,2-dihexanoyl- sn-glycero-3-phophocholine (DHPC) (4:1) and DMPC/1,2-dimyristoyl- sn-glycero-3-phopho-(1'-rac-glycerol) (DMPG)/DHPC (3:1:1), two mixtures of long-chain and short-chain lipids that display bicellar behavior. This study exploited the sensitivity of a bicellar system structural organization to factors that modify partitioning of their lipid components between different environments. In small bicelle particles formed at low temperatures, short-chain components preferentially occupy curved rim environments around bilayer disks of the long-chain components. Changes in chain order and lipid mixing, on heating, can drive transitions to more extended assemblies including a magnetically orientable phase at intermediate temperature. In this work, neither peptide had a substantial effect on the behavior of the zwitterionic DMPC/DHPC mixture. For bicellar mixtures containing the anionic lipid DMPG, the peptide SP-B63-78 lowered the temperature at which magnetically orientable particles coalesced into more extended lamellar structures. SP-B63-78 did not promote partitioning of the zwitterionic and anionic long-chain lipid components into different environments. Magainin 2, on the other hand, was found to promote separation of the anionic lipid, DMPG, and the zwitterionic lipid, DMPC, into different environments for temperatures above 34 °C. The contrast between the effects of these two peptides on the lipid mixtures studied appears to be consistent with their functional roles in biological systems.

Characterization of a peptide containing the major heparin binding domain of human hepatic lipase.

Human hepatic lipase (hHL) is a cell surface associated enzyme that hydrolyzes triacylglycerols and phospholipids within circulating lipoproteins. We hypothesized that an amino acid sequence mimicking the major heparin binding domain (HBD) of hHL will displace hHL from cell surfaces. To test this hypothesis, we generated a recombinant protein of thioredoxin linked with a cleavable, tagged sequence containing amino acids 442 to 476 of the mature hHL sequence, which contains the major HBD of hHL. The recombinant protein associated with heparin-sepharose, and its peak elution from heparin-sepharose occurred in the presence of 0.5 M NaCl. We cleaved and purified the tagged sequence containing the HBD from the recombinant protein and tested the ability of the peptide to displace full-length hHL from HEK-293 cells. The peptide indeed displaced hHL from cell surfaces, while no significant displacement was observed in the presence of a peptide with a scrambled sequence. Finally, we obtained structural information for the peptide containing the HBD. 1 H- and 15 N-NMR spectra of the peptide indicate the peptide is largely unstructured, although not completely random coil. The addition of heparin to the peptide induced some changes in chemical shift, suggesting changes in peptide structure and/or specific interactions with heparin. Molecular simulations confirm the largely unstructured nature of the isolated peptide, but they also indicate weak tendencies for both α- and ß-structure formation in different parts of the chain. Overall, these data provide a proof-of-principle for the use of mimetic peptides for the displacement of cell surface associated lipases.

All-atom molecular dynamics simulations of lung surfactant protein B: Structural features of SP-B promote lipid reorganization.

Lung surfactant protein B (SP-B), a 79 residue, hydrophobic protein from the saposin superfamily, plays an essential role in breathing. Because of the extreme hydrophobicity of SP-B, the experimental structure of this protein has not yet been determined. Here, we run all-atom molecular dynamics simulations using the OPLS-AA force field in GROMACS to study SP-B's structure and mechanisms for promoting lipid reorganization. Firstly, we find that the final structures indicate the need for some fine-tuning of the homology-based secondary structure predictions. Secondly, we find energetically feasible structures 1) with SP-B's helices in the plane of the bilayer, 2) with SP-B's helices inclined with respect to the bilayer, and 3) with SP-B in a closed structure interacting peripherally with the bilayer. Interestingly, SP-B structures that were bent at the hinge region between the pairs of helices promoted and/or stabilized defects in the lipid bilayer. Finally, particular salt bridge patterns and structural plasticity in the central loop and adjacent region of SP-B appeared to be involved in SP-B's lipid reorganization abilities.

Recent progress on the application of 2H solid-state NMR to probe the interaction of antimicrobial peptides with intact bacteria.

Discoveries relating to innate immunity and antimicrobial peptides (AMPs) granted Bruce Beutler and Jules Hoffmann a Nobel prize in medicine in 2011, and opened up new avenues for the development of therapies against infections, and even cancers. The mechanisms by which AMPs interact with, and ultimately disrupt, bacterial cell membranes is still, to a large extent, incompletely understood. Up until recently, this mechanism was studied using model lipid membranes that failed to reproduce the complexity of molecular interactions present in real cells comprising lipids but also membrane proteins, a cell wall containing peptidoglycan or lipopolysaccharides, and other molecules. In this review, we focus on recent attempts to study, at the molecular level, the interaction between cationic AMPs and intact bacteria, by 2H solid-state NMR. Specifically-labeled lipids allow us to focus on the interaction of AMPs with the heart of the bacterial membrane, and measure the lipid order and its variation upon interaction with various peptides. We will review the important parameters to consider in such a study, and summarize the results obtained in the past 5years on various peptides, in particular aurein 1.2, caerin 1.1, MSI-78 and CA(1-8)M(1-10). This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Structure-function relationships in histidine-rich antimicrobial peptides from Atlantic cod.

Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure-function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2's percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2's highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.

Molecular dynamics simulations of histidine-containing cod antimicrobial peptide paralogs in self-assembled bilayers.

Gaduscidin-1 and -2 (GAD-1 and GAD-2) are antimicrobial peptides (AMPs) that contain several histidine residues and are thus expected to exhibit pH-dependent activity. In order to help elucidate their mechanism of membrane disruption, we have performed molecular dynamics simulations with the peptides in both histidine-charged and histidine-neutral forms, along with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid molecules. The simulations employed GROMACS software and an OPLS-AA force field. Initially, the peptide and lipids were placed randomly in the simulation box and then were allowed to self-assemble. The results demonstrated a marked preference for the regions of the peptides that contain sequential pairs of histidine residues to associate closely with bilayer pores. This preference is observed even when the histidines are in their uncharged form. It appears that the relative compactness and rigidity of histidine pairs require the more aqueous and disordered environment of the pores to satisfy hydrophilic interactions. The final peptide structures exhibited a wide variety of structures and topologies, with the most helical structures positioning most parallel to the bilayer surface and the less ordered structures interacting more closely with the pore. Thus, the results give atomistic insight into those models of AMP mechanism that promote the importance of structural heterogeneity and imperfect amphipathicity to AMP activity and selectivity.

Recurrent missense mutations in TMEM43 (ARVD5) due to founder effects cause arrhythmogenic cardiomyopathies in the UK and Canada.

AIMS: Autosomal dominant arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) (in the group of arrhythmogenic cardiomyopathies) is a common cause of sudden cardiac death in young adults. It is both clinically and genetically heterogeneous, with 12 loci (ARVC/D1-12) and eight genes identified, the majority of which encode structural proteins of cardiac desmosomes. The most recent gene identified, TMEM43, causes disease due to a missense mutation in a non-desmosomal gene (p.S358L) in 15 extended families from Newfoundland, Canada. To determine whether mutations in TMEM43 cause ARVC/D and arrhythmogenic cardiomyopathy in other populations, we fully re-sequenced TMEM43 on 143 ARVC/D probands (families) from the UK and 55 probands (from 55 families) from Newfoundland. METHODS AND RESULTS: Bidirectional sequencing of TMEM43 including intron-exon boundaries revealed 33 variants, the majority located in non-coding regions of TMEM43. For the purpose of validation, families of probands with rare, potentially deleterious coding variants were subjected to clinical and molecular follow-up. Three missense variants of uncertain significance (p.R28W, p.E142K, p.R312W) were located in highly conserved regions of the TMEM43 protein. One variant (p.R312W) also co-segregated with relatives showing clinical signs of disease. Genotyping and expansion of the disease-associated haplotype in subjects with the p.R312W variant from Newfoundland, Canada, and the UK suggest common ancestry. CONCLUSION: Although the p.R312W variant was found in controls (3/378), identification of an ancestral disease p R312W haplotype suggests that the p.R312W variant is a pathogenic founder mutation.

The effects of biological crowders on fibrillization, structure, diffusion, and conformational dynamics of α-synuclein.

α-synuclein is an intrinsically disordered protein (IDP) whose aggregation in presynaptic neuronal cells is a pathological hallmark of Lewy body formation and Parkinson's disease. This aggregation process is likely affected by the crowded macromolecular cellular environment. In this study, α-synuclein was studied in the presence of both a synthetic crowder, Ficoll70, and a biological crowder composed of lysed cells that better mimics the biocomplexity of the cellular environment. 15 N-1 H HSQC NMR results show similar α-synuclein chemical shifts in non-crowded and all crowded conditions implying that it remains similarly unstructured in all conditions. Nevertheless, both HSQC NMR and fluorescence measurements indicate that, only in the cell lysate, α-synuclein forms aggregates over a timescale of 48 h. 15 N-edited diffusion measurements indicate that all crowders slow down the α-synuclein's diffusivity. Interestingly, at high concentrations, α-synuclein diffuses faster in cell lysate than in Ficoll70, possibly due to additional soft (e.g., electrostatic or hydrophobic) interactions. 15 N-edited relaxation measurements show that some residues are more mobile in cell lysate than in Ficoll70; the rates that are most different are predominantly in hydrophobic residues. We thus examined cell lysates with reduced hydrophobicity and found slower dynamics (higher relaxation rates) in several α-synuclein residues. Taken together, these experiments suggest that while cell lysate does not substantially affect α-synuclein structure (HSQC spectra), it does affect chain dynamics and translational diffusion, and strongly affects aggregation over a timescale of days, in a manner that is different from either no crowder or an artificial crowder: soft hydrophobic interactions are implicated.

Orientation and depth of surfactant protein B C-terminal helix in lung surfactant bilayers.

SP-B(CTERM) is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B(CTERM) exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B(CTERM)-induced lipid realignment and/or conformational changes at the level of the head group were observed using (31)P solid-state NMR spectroscopy. Measurements of the depth of SP-B(CTERM) indicated the peptide center positions ~8Å more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression.

Diffusion NMR study of complex formation in membrane-associated peptides.

Pulsed-field-gradient nuclear magnetic resonance (PFG-NMR) is used to obtain the true hydrodynamic size of complexes of peptides with sodium dodecyl sulfate SDS micelles. The peptide used in this study is a 19-residue antimicrobial peptide, GAD-2. Two smaller dipeptides, alanine-glycine (Ala-Gly) and tyrosine-leucine (Tyr-Leu), are used for comparison. We use PFG-NMR to simultaneously measure diffusion coefficients of both peptide and surfactant. These two inputs, as a function of SDS concentration, are then fit to a simple two species model that neglects hydrodynamic interactions between complexes. From this we obtain the fraction of free SDS, and the hydrodynamic size of complexes in a GAD-2-SDS system as a function of SDS concentration. These results are compared to those for smaller dipeptides and for peptide-free solutions. At low SDS concentrations ([SDS] ≤ 25 mM), the results self-consistently point to a GAD-2-SDS complex of fixed hydrodynamic size R = (5.5 ± 0.3) nm. At intermediate SDS concentrations (25 mM < [SDS] < 60 mM), the apparent size of a GAD-2-SDS complex shows almost a factor of two increase without a significant change in surfactant-to-peptide ratio within a complex, most likely implying an increase in the number of peptides in a complex. For peptide-free solutions, the self-diffusion coefficients of SDS with and without buffer are significantly different at low SDS concentrations but merge above [SDS] = 60 mM. We find that in order to obtain unambiguous information about the hydrodynamic size of a peptide-surfactant complex from diffusion measurements, experiments must be carried out at or below [SDS] = 25 mM.

²H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78.

A key aspect of the activity of antimicrobial peptides (AMPs) is their interaction with membranes. Efforts to elucidate their detailed mechanisms have focused on applying biophysical methods, including nuclear magnetic resonance (NMR), to AMPs in model lipid systems. However, these highly simplified systems fail to capture many of the features of the much more complex cell envelopes with which AMPs interact in vivo. To address this issue, we have designed a procedure to incorporate high levels of (2)H NMR labels specifically into the cell membrane of Escherichia coli and used this approach to study the interactions between the AMP MSI-78 and the membranes of intact bacteria. The (2)H NMR spectra of these membrane-deuterated bacteria can be reproduced in the absence and presence of MSI-78. Because the (2)H NMR data provide a quantitative measure of lipid disorder, they directly report on the lipid bilayer disruption central to the function of AMPs, in the context of intact bacteria. Addition of MSI-78 to the bacteria leads to decreases in the order of the lipid acyl chains. The molar peptide:lipid ratios required to observe the effects of MSI-78 on acyl chain order are approximately 30 times greater than the ratios needed to observe effects in model lipid systems and approximately 100 times less than the ratios required to observe inhibition of cell growth in biological assays. The observations thus suggest that MSI-78 disrupts the bilayer even at sublethal AMP levels and that a large fraction of the peptide does not actually reach the inner membrane.

Effects of the lung surfactant protein B construct Mini-B on lipid bilayer order and topography.

The hydrophobic lung surfactant protein, SP-B, is essential for survival. Cycling of lung volume during respiration requires a surface-active lipid-protein layer at the alveolar air-water interface. SP-B may contribute to surfactant layer maintenance and renewal by facilitating contact and transfer between the surface layer and bilayer reservoirs of surfactant material. However, only small effects of SP-B on phospholipid orientational order in model systems have been reported. In this study, N-terminal (SP-B(8-25)) and C-terminal (SP-B(63-78)) helices of SP-B, either linked as Mini-B or unlinked but present in equal amounts, were incorporated into either model phospholipid mixtures or into bovine lipid extract surfactant in the form of vesicle dispersions or mechanically oriented bilayer samples. Deuterium and phosphorus nuclear magnetic resonance (NMR) were used to characterize effects of these peptides on phospholipid chain orientational order, headgroup orientation, and the response of lipid-peptide mixtures to mechanical orientation by mica plates. Only small effects on chain orientational order or headgroup orientation, in either vesicle or mechanically oriented samples, were seen. In mechanically constrained samples, however, Mini-B and its component helices did have specific effects on the propensity of lipid-peptide mixtures to form unoriented bilayer populations which do not exchange with the oriented fraction on the timescale of the NMR experiment. Modification of local bilayer orientation, even in the presence of mechanical constraint, may be relevant to the transfer of material from bilayer reservoirs to a flat surface-active layer, a process that likely requires contact facilitated by the formation of highly curved protrusions.

Conjugates for use in peptide therapeutics: A systematic review and meta-analysis.

While peptides can be excellent therapeutics for several conditions, their limited in vivo half-lives have been a major bottleneck in the development of therapeutic peptides. Conjugating the peptide to an inert chemical moiety is a strategy that has repeatedly proven to be successful in extending the half-life of some therapeutics. This systematic review and meta-analysis was conducted to examine the available literature and assess it in an unbiased manner to determine which conjugates, both biological and synthetic, provide the greatest increase in therapeutic peptide half-life. Systematic searches run on PubMed, Scopus and SciFinder databases resulted in 845 studies pertaining to the topic, 16 of these were included in this review after assessment against pre-specified inclusion criteria registered on PROSPERO (#CRD42020222579). The most common reasons for exclusion were non-IV administration and large peptide size. Of the 16 studies that were included, a diverse suite of conjugates that increased half-life from 0.1 h to 33.57 h was identified. Amongst these peptides, the largest increase in half-life was seen when conjugated with glycosaminoglycans. A meta-analysis of studies that contained fatty acid conjugates indicated that acylation contributed to a statistically significant extension of half-life. Additionally, another meta-analysis followed by a sensitivity analysis suggested that conjugation with specifically engineered recombinant peptides might contribute to a more efficient extension of peptide half-life as compared to PEGylation. Moreover, we confirmed that while polyethylene glycol is a good synthetic conjugate, its chain length likely has an impact on its effectiveness in extending half-life. Furthermore, we found that most animal studies do not include as much detail when reporting findings as compared to human studies. Inclusion of additional experimental detail on aspects such as independent assessment and randomization may be an easily accomplished strategy to drive more conjugated peptides towards clinical studies.

Role of lipopolysaccharide in antimicrobial and cell penetrating peptide membrane interactions probed by deuterium NMR of whole cells.

Understanding how non-lipid components of bacteria affect antimicrobial peptide (AMP)-induced membrane disruption is important for a comprehensive understanding of AMP mechanisms and informing AMP-based drug development. This study investigates how lipopolysaccharide (LPS) affects membrane disruption by the AMP MSI-78 and compares the results to the effect of TP2, a cell-penetrating peptide that crosses membrane bilayers without permeabilizing them. We destabilize the LPS layer of Escherichia coli (E. coli) cells via chelation of the stabilizing divalent cations. 2H NMR spectra of E. coli demonstrate that EDTA concentrations of 2.5 mM and 9.0 mM alone have very minor effects on lipid acyl chain order. Interestingly, we find that E. coli pre-treated with 9.0 mM EDTA before treatment with MSI-78 are more sensitive to AMP-induced acyl chain disruption, indicating that intact LPS reduces MSI-78-induced membrane disruption in E. coli. Surprisingly, we also found that at the level of 2H_NMR, the peptide-induced acyl chain disruption is similar for MSI-78 and TP2, although MSI-78 permeabilizes the bilayer and TP2 does not. Furthermore, LPS disruption appears to protect the bacteria from TP2, although it sensitizes them to MSI-78.

Interaction between Antimicrobial Peptide Magainin 2 and Nonlipid Components in the Bacterial Outer Envelope.

Antimicrobial peptides (AMPs) offer advantages over conventional antibiotics; for example, bacteria develop more resistance to small-molecule antibiotics than to AMPs. The interaction of the AMPs with the lipopolysaccharide (LPS) layer of the Gram-negative bacteria cell envelope is not well understood. A MARTINI model was constructed of a Gram-negative bacterial outer membrane interacting with the AMP Magainin 2. In a 20 µs molecular dynamics (MD) simulation, the AMP diffused to the LPS layer of the cell envelope and remained there, suggesting interactions between the Magainin 2 and the LPS layer, causing the AMP to concentrate at that position. The free energy profile for the insertion of the Magainin 2 into the membrane was also calculated using umbrella sampling, which showed that the AMP positioned such that the cationic side chains of the AMP coordinated to the negatively charged phosphate groups of the LPS layer. These simulations indicate that the AMP Magainin 2 partition into the LPS layer of a bacterial membrane.