NMR methods for studying membrane‐active antimicrobial peptides

Strandberg, Erik; Ulrich, Anne S.

doi:10.1002/cmr.a.20024

Cited by 142 publications

(124 citation statements)

References 243 publications

Supporting

Mentioning

121

Contrasting

Order By: Relevance

“…In both cases, the software predictions for the almost central residue G8 had a low consensus (and were not included in Figure 1), suggesting a flexible kink that divides the peptide sequence into two helical fragments. However, in the case of pure DPC micelles, the residue S4, which is exactly in the middle of the N-terminal fragment, was predicted to have / values not compatible with an -helix (Figure 1c), thus suggesting a regular secondary structure only for the C-terminal fragment. Figure 2 summarizes the sequential NOEs unambiguously identified for Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) in the presence of either DPC or DPC/SDS micelles. Figure S1 show the H-HN region of the NOESY spectra.…”

Section: Resultsmentioning

confidence: 99%

“…This particular DPC/SDS molar ratio was chosen to be close to that between zwitterionic and negatively charged lipids typical for the inner membrane of Gram-negative bacteria. 25 Table S1 reports the 1 H and 13 C resonance assignments for Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) in the presence of either DPC or DPC/SDS micelles, obtained through a set of two-dimensional homo-and heteronuclear experiments, namely, DQF-COSY, TOCSY, NOESY, and HSQC. Figures 1a and 1b show the deviations of the observed chemical shifts from the reference "random coil" values for 1 H and 13 C, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…8 Several recent articles have reviewed in detail the different modes of action postulated for AMPs. [9][10][11] The amphipathic conformation adopted by the AMP, with the hydrophilic residues interacting with the polar head groups of the phospholipids and the water molecules, and the hydrophobic residues being protected from water contacts by inserting into the lipid bilayer core, is considered essential for it to exert the antimicrobial activity. Also the net positive charge plays a fundamental role.…”

mentioning

confidence: 99%

“…23 A globally amphipathic structure was found, with a helical conformation for the N-terminal portion (encompassing the first 12 residues) and an unfolded C-terminal fragment. 23 However, as the H 2 O/TFE solvent is an isotropic environment, such a structural model for Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) should be taken with caution. Despite promoting peptide folding by favoring intramolecular H-bonds, indeed, H 2 O/TFE mixtures inherently lack the intrinsic anisotropic character of a lipid bilayer.…”

mentioning

confidence: 99%

“…In particular, both zwitterionic and negatively charged micelles have been employed to model eukaryotic and prokaryotic plasma membrane, respectively, as bacterial membranes are usually characterized by a higher content of negatively charged lipids than the former. 6,12,13 The insertion depth of Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) into the micelles was estimated by using Mn 2+ ions as a paramagnetic probe. The results clearly confirm the helical folding of Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and show how binding and folding are prominent only in the presence of a negatively charged membrane.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Folded Structure and Insertion Depth of the Frog-Skin Antimicrobial Peptide Esculentin-1b(1–18) in the Presence of Differently Charged Membrane-Mimicking Micelles

Manzo¹,

Casu²,

Rinaldi³

et al. 2014

J. Nat. Prod.

View full text Add to dashboard Cite

Antimicrobial peptides (AMPs) are effectors of the innate immunity of most organisms. Their role in the defense against pathogen attack and their high selectivity for bacterial cells make them attractive for the development of a new class of antimicrobial drugs. The N-terminal fragment of the frog-skin peptide esculentin-1b (Esc(1-18)) has shown broad spectrum antimicrobial activity.Similarly to most cationic AMPs, it is supposed to act by binding to and damaging the negatively charged plasma-membrane of bacteria. Differently from many other AMPs, Esc(1-18) activity is preserved in biological fluids such as serum. In this work, a structural investigation was performed through NMR spectroscopy. The 3D structure was obtained in the presence of either zwitterionic or negatively charged micelles as membrane models for eukaryotic and prokaryotic membranes, respectively. Esc(1-18) showed a higher affinity for and deeper insertion into the latter, and adopted an amphipathic helical structure characterized by a kink at the residue G8. These findings were confirmed by measuring penetration into lipid monolayers. The presence of negatively charged lipids in the bilayer appears to be necessary for Esc(1-18) to bind, fold in the right threedimensional structure and, ultimately, to exert its biological role as an AMP. 3The lack of a new class of antibiotics is an urgent problem, as many important pathogens are rapidly developing resistance to most of the clinically usable antibiotics. 1,2 Clinical impact of antibiotic resistance is immense, with increasing length of hospital stay, costs and mortality. 3,4 Antimicrobial peptides (AMPs) have attracted the interest of the scientific community as promising candidates for the development of a new class of antimicrobial drugs. They are part of the innate immune system of the majority of the multicellular organisms, and are typically characterized by a wide spectrum of antimicrobial activity, from Gram-negative to Gram-positive bacteria, fungi, protozoa and enveloped viruses. [5][6][7] Despite amino acid sequence homology is not high, AMPs share some common features. They are relatively short (< 60 residues), cationic, have a large content (≥ 50%) of hydrophobic residues and adopt an amphipathic three-dimensional folding when interacting with pathogens" membranes. 6 Generally, AMPs destroy or permeate the microbial plasma-membrane. The membrane is formed through evolutionarily well conserved biosynthetic processes and represents a non-selective target, making modifications by the microorganisms difficult and, thus, resistance onset is unlikely. 8 Several recent articles have reviewed in detail the different modes of action postulated for AMPs. [9][10][11] The amphipathic conformation adopted by the AMP, with the hydrophilic residues interacting with the polar head groups of the phospholipids and the water molecules, and the hydrophobic residues being protected from water contacts by inserting into the lipid bilayer core, is considered essential for it to exert the antimicro...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Folded Structure and Insertion Depth of the Frog-Skin Antimicrobial Peptide Esculentin-1b(1–18) in the Presence of Differently Charged Membrane-Mimicking Micelles

Manzo¹,

Casu²,

Rinaldi³

et al. 2014

J. Nat. Prod.

View full text Add to dashboard Cite

show abstract

Cell Membrane Penetration without Pore Formation: Chameleonic Properties of Dendrimers in Response to Hydrophobic and Hydrophilic Environments

Luca

Seal

Parekh

et al. 2020

Advcd Theory and Sims

View full text Add to dashboard Cite

The mechanism by which cell-penetrating peptides and antimicrobial peptides cross plasma membranes is unknown, as is how cell-penetrating peptides facilitate drug delivery, mediating the transport of small molecules. Once non-disruptive and non-endocytotic pathways are excluded, pore formation is one of the proposed mechanisms, including toroidal, barrel-stave, or carpet models.Spontaneous pores have been observed in coarse-grained simulations and less often in molecular dynamics simulations. While pores are widely assumed and inferred, there is no unambiguous Antibiotic-resistant bacteria constitute a significant public health problem 1,2 . Antimicrobial peptides (AMPs) -a class of small (≤50 residues), cationic or amphiphilic compounds -are amongst the most promising solutions currently known 3 . Cell-penetrating peptides (CPPs) are a similar class of peptides, important for their binding -usually via covalent conjugation through a disulfide bridge or by means of non-covalent interactions 4 -and delivery of drugs into cells for therapeutic applications [5][6][7] . Both CPPs and AMPs interact strongly with the membrane surface, penetrating or altering its permeability via a mechanism which is not well understood 8,9 , even after a plethora of experimental and simulation studies.Atomistic molecular dynamics (MD) can be applied to investigate certain aspects of the uptake mechanisms. Despite its limitations in terms of accessible spatial and temporal scales, MD has the potential to provide unique insights. Several studies have converged, via observations or deductions, to infer the formation or existence of pores in the membrane 10-14 . Pores have been observed in coarse-grained simulations 15,16 , and in molecular dynamics 17,18 . A spontaneous translocation of a TAT peptide was observed in one MD study 19 and explained in terms of pore formation. However, subsequently this result was found to have originated from incorrect use of electrostatic interactions 14 . When a phosphatidylcholine (POPC) membrane is exposed to a CPP such as maculatin 1.1, dye leakage was observed after ∼10 minutes. This result led the authors of the study to infer a physical mechanism based on membrane disruption or pore formation 20 .Binding energies of pore formation with alamethicin and melittin showed preference for cylindrical 21 and toroidal 22 pores, in accord with molecular dynamics results 23 .In contrast to the above picture, some experiments do not support the presence of pores.Considering that cationic peptides should interact more strongly with negatively charged compounds, one would have expected pore formations with a CPP oligoarginine peptide interacting with negatively charged lipids. Interestingly, a leakage was observed, but not with an ion-conductive structure 24 , which would have been expected in presence of holes or channels.Trans-activating transcriptional (TAT) peptide penetration has been observed in the absence of endocytosis, with the additional discovery that added fluorophores were not taken together with the...

show abstract

Toward an improved structural model of the frog‐skin antimicrobial peptide esculentin‐1b(1‐18)

Manzo¹,

Sanna²,

Casu³

et al. 2012

Biopolymers

View full text Add to dashboard Cite

Antimicrobial peptides (AMPs) are found in various classes of organisms as part of the innate immune system. Despite high sequence variability, they share common features such as net positive charge and an amphipathic fold when interacting with biologic membranes. Esculentin-1b is a 46-mer frog-skin peptide, which shows an outstanding antimicrobial activity. Experimental studies revealed that the N-terminal fragment encompassing the first 18 residues, Esc(1-18), is responsible for the antimicrobial activity of the whole peptide, with a negligible toxicity toward eukaryotic cells, thus representing an excellent candidate for future pharmaceutical applications. Similarly to most of the known AMPs, Esc(1-18) is expected to act by destroying/permeating the bacterial plasma-membrane but, to date, its 3D structure and the detailed mode of action remains unexplored. Before an in-depth investigation on peptide/membranes interactions could be undertaken, it is necessary to characterize peptide's folding propensity in solution, to understand what is intrinsically due to the peptide sequence, and what is actually driven by the membrane interaction. Circular dichroism and nuclear magnetic resonance spectroscopy were used to determine the structure adopted by the peptide, moving from water to increasing amounts of trifluoroethanol. The results showed that Esc(1-18) has a clear tendency to fold in a helical conformation as hydrophobicity of the environment increases, revealing an intriguing amphipathic structure. The helical folding is adopted only by the N-terminal portion of the peptide, while the rest is unstructured. The presence of a hydrophobic cluster of residues in the C-terminal portion suggests its possible membrane-anchoring role.

show abstract

NMR methods for studying membrane‐active antimicrobial peptides

Cited by 142 publications

References 243 publications

Folded Structure and Insertion Depth of the Frog-Skin Antimicrobial Peptide Esculentin-1b(1–18) in the Presence of Differently Charged Membrane-Mimicking Micelles

Folded Structure and Insertion Depth of the Frog-Skin Antimicrobial Peptide Esculentin-1b(1–18) in the Presence of Differently Charged Membrane-Mimicking Micelles

Cell Membrane Penetration without Pore Formation: Chameleonic Properties of Dendrimers in Response to Hydrophobic and Hydrophilic Environments

Toward an improved structural model of the frog‐skin antimicrobial peptide esculentin‐1b(1‐18)

Contact Info

Product

Resources

About