NMR techniques for investigating antimicrobial peptides in model membranes and bacterial cells.

AMPs are short, mainly cationic membrane-active peptides found in all living organism. They perform diverse roles including signaling and acting as a line of defense against bacterial infections. AMPs have been extensively investigated as templates to facilitate the development of novel antimicrobial therapeutics. Understanding the interplay between these membrane-active peptides and the lipid membranes is considered to be a significant step in elucidating the specific mechanism of action of AMPs against prokaryotic and eukaryotic cells to aid the development of new therapeutics. In this review, we have provided a brief overview of various NMR techniques commonly used for studying AMP structure and AMP-membrane interactions in model membranes and whole cells.

The membrane activity of the antimicrobial peptide caerin 1.1 is pH dependent.

Antimicrobial peptides are an important class of membrane-active peptides that can provide alternatives or complements to classic antibiotics. Among the many classes of AMPs, the histidine-rich family is of particular interest since they may induce pH-sensitive interactions with cell membranes. The AMP caerin 1.1 (Cae-1), from Australian tree frogs, has three histidine residues, and thus we studied the pH dependence of its interactions with model cell membranes. Using NMR spectroscopy and molecular dynamics simulations, we showed that Cae-1 induced greater perturbation of the lipid dynamics and water penetrations within the membrane interior in an acidic environment compared with physiological conditions. Using 31P solid-state NMR, the packing, chemical environment, and dynamics of the lipid headgroup were monitored. 2H solid-state NMR showed that Cae-1 ordered the acyl chains of the hydrophobic core of the bilayer. These results supported the molecular dynamics data, which showed that Cae-1 was mainly inserted within the lipid bilayer for both neutral and negatively charged membranes, with the charged residues pulling the water and phosphate groups inward. This could be an early step in the mechanism of membrane disruption by histidine-rich antimicrobial peptides and indicated that Cae-1 acts via a transmembrane mechanism in bilayers of neutral and anionic phospholipid membranes, especially in acidic conditions.

Ultrasound-induced protein restructuring and ordered aggregation to form amyloid crystals.

Amyloid crystals, a form of ordered protein aggregates documented relatively recently, have not been studied as extensively as amyloid fibres. This study investigates the formation of amyloid crystals with low frequency ultrasound (20 kHz) using ß-lactoglobulin, as a model protein for amyloid synthesis. Acoustic cavitation generates localised zones of intense shear, with extreme heat and pressure that could potentially drive the formation of amyloid structures at ambient bulk fluid temperatures (20 ± 1 °C). Thioflavin T fluorescence and electron microscopy showed that low-frequency ultrasound at 20 W/cm3 input power induced ß-stacking to produce amyloid crystals in the mesoscopic size range, with a mean length of approximately 22 µm. FTIR spectroscopy indicated a shift towards increased intermolecular antiparallel ß-sheet content. An increase in sonication time (0-60 min) and input power (4-24 W/cm3) increased the mean crystal length, but this increase was not linearly proportional to sonication time and input power due to the delayed onset of crystal growth. We propose that acoustic cavitation causes protein unfolding and aggregation and imparts energy to aggregates to cross the torsion barrier, to achieve their lowest energy state as amyloid crystals. The study contributes to a further understanding of protein chemistry relating to the energy landscape of folding and aggregation. Ultrasound presents opportunities for practical applications of amyloid structures, presenting a more adaptable and scalable approach for synthesis.

NMR measurement of biomolecular translational and rotational motion for evaluating changes of protein oligomeric state in solution.

Defining protein oligomeric state and/or its changes in solution is of significant interest for many biophysical studies carried out in vitro, especially when the nature of the oligomeric state is crucial in the subsequent interpretation of experimental results and their biological relevance. Nuclear magnetic resonance (NMR) is a well-established methodology for the characterization of protein structure, dynamics, and interactions at the atomic level. As a spectroscopic method, NMR also provides a compelling means for probing both molecular translational and rotational motion, two predominant measures of effective molecular size in solution, under identical conditions as employed for structural, dynamic and interaction studies. Protein translational diffusion is readily measurable by pulse gradient spin echo (PGSE) NMR, whereas its rotational correlation time, or rotational diffusion tensor when its 3D structure is known, can also be quantified from NMR relaxation parameters, such as 15N relaxation parameters of backbone amides which are frequently employed for probing residue-specific protein backbone dynamics. In this article, we present an introductory overview to the NMR measurement of bimolecular translational and rotational motion for assessing changes of protein oligomeric state in aqueous solution, via translational diffusion coefficients measured by PGSE NMR and rotational correlation times derived from composite 15N relaxation parameters of backbone amides, without need for the protein structure being available.

Transformation of L-DOPA and Dopamine on the Surface of Gold Nanoparticles: An NMR and Computational Study.

Gold nanoparticles (AuNPs) have found applications in biomedicine as diagnostic tools, but extensive research efforts have been also directed toward their development as more efficient drug delivery agents. The high specific surface area of AuNPs may provide dense loading of molecules like catechols (L-DOPA and dopamine) on nanosurfaces, enabling functionalization strategies for advancing conventional therapy and diagnostic approaches of neurodegenerative diseases. Despite numerous well-described procedures in the literature for preparation of different AuNPs, possible transformation and structural changes of surface functionalization agents have not been considered thoroughly. As a case in point, the catechols L-DOPA and dopamine were selected because of their susceptibility to oxidation, cyclization, and polymerization. To assess the fate of coating and functionalization agents during the preparation of AuNPs or interaction at the nano-bio interface, a combination of spectroscopy, light scattering, and microscopy techniques was used while structural information and reaction mechanism were obtained by NMR in combination with computational tools. The results revealed that the final form of catechol on the AuNP nanosurface depends on the molar ratio of Au used for AuNP preparation. A large molar excess of L-DOPA or dopamine is needed to prepare AuNPs funtionalized with fully reduced catechols. In the case of molar excess of Au, the oxidation of catechols to dopamine quinone and dopaquinone was promoted, and dopaquinone underwent intramolecular cyclization in which additional oxidation products, leukodopachrome, dopachrome, or its tautomer, were formed because of the larger intrinsic acidity of the more nucleophilic amino group in dopaquinone. MD simulations showed that, of the oxidation products, dopachrome had the highest affinity for binding to the AuNPs surface. The results highlight how a more versatile methodological approach, combining experimental and in silico techniques, allows more reliable characterization of binding events at the surface of AuNPs for possible applications in biomedicine.

Chemically modified and conjugated antimicrobial peptides against superbugs.

Antimicrobial resistance (AMR) is one of the greatest threats to human health that, by 2050, will lead to more deaths from bacterial infections than cancer. New antimicrobial agents, both broad-spectrum and selective, that do not induce AMR are urgently required. Antimicrobial peptides (AMPs) are a novel class of alternatives that possess potent activity against a wide range of Gram-negative and positive bacteria with little or no capacity to induce AMR. This has stimulated substantial chemical development of novel peptide-based antibiotics possessing improved therapeutic index. This review summarises recent synthetic efforts and their impact on analogue design as well as their various applications in AMP development. It includes modifications that have been reported to enhance antimicrobial activity including lipidation, glycosylation and multimerization through to the broad application of novel bio-orthogonal chemistry, as well as perspectives on the direction of future research. The subject area is primarily the development of next-generation antimicrobial agents through selective, rational chemical modification of AMPs. The review further serves as a guide toward the most promising directions in this field to stimulate broad scientific attention, and will lead to new, effective and selective solutions for the several biomedical challenges to which antimicrobial peptidomimetics are being applied.

TOAC spin-labeled peptides tailored for DNP-NMR studies in lipid membrane environments.

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better 13C and 15N signal NMR enhancements and 1H spin-lattice T1 relaxation than T-MacW.

Utilizing magnetic resonance techniques to study membrane interactions of amyloid peptides.

Alzheimer's disease (AD) is a common neurodegenerative condition that involves the extracellular accumulation of amyloid plaques predominantly consisting of Aß peptide aggregates. The amyloid plaques and soluble oligomeric species of Aß are believed to be the major cause of synaptic dysfunction in AD brain and their cytotoxic mechanisms have been proposed to involve interactions with cell membranes. In this review, we discuss our solid-state nuclear magnetic resonance (ssNMR) studies of Aß interactions with model membranes.

C-terminus amidation influences biological activity and membrane interaction of maculatin 1.1.

Cationic antimicrobial peptides have been investigated for their potential use in combating infections by targeting the cell membrane of microbes. Their unique chemical structure has been investigated to understand their mode of action and optimize their dose-response by rationale design. One common feature among cationic AMPs is an amidated C-terminus that provides greater stability against in vivo degradation. This chemical modification also likely modulates the interaction with the cell membrane of bacteria yet few studies have been performed comparing the effect of the capping groups. We used maculatin 1.1 (Mac1) to assess the role of the capping groups in modulating the peptide bacterial efficiency, stability and interactions with lipid membranes. Circular dichroism results showed that C-terminus amidation maintains the structural stability of the peptide (α-helix) in contact with micelles. Dye leakage experiments revealed that amidation of the C-terminus resulted in higher membrane disruptive ability while bacteria and cell viability assays revealed that the amidated form displayed higher antibacterial ability and cytotoxicity compared to the acidic form of Mac1. Furthermore, 31P and 2H solid-state NMR showed that C-terminus amidation played a greater role in disturbance of the phospholipid headgroup but had little effect on the lipid tails. This study paves the way to better understand how membrane-active AMPs act in live bacteria.

The impact of antibacterial peptides on bacterial lipid membranes depends on stage of growth.

The impact of maculatin 1.1 (Mac1) on the mechanical properties of supported lipid membranes derived from exponential growth phase (EGP) and stationary growth phase (SGP) E. coli lipid extracts was analysed by surface plasmon resonance and atomic force microscopy. Each membrane was analysed by quantitative nanomechanical mapping to derive measurements of the modulus, adhesion and deformation in addition to bilayer height. Image analysis revealed the presence of two domains in the EGP membrane differing in height by 0.4 nm. Three distinct domains were observed in the SGP membrane corresponding to 4.2, 4.7 and 5.4 nm in height. Using surface plasmon resonance, Mac1 was observed to bind strongly to both membranes and then disrupt the membranes as evidenced by a sharp drop in baseline. Atomic force microscopy (AFM) topographic analysis revealed the formation of domains of different height and confirmed that membrane destruction was much faster for the SGP derived bilayer. Moreover, Mac1 selectively disrupted the domain with the lowest thickness, which may correspond to a liquid ordered domain. Overall, the results provide insight into the role of lipid domains in the response of bacteria to antimicrobial peptides.

Cholesterol-Dependent Cytolysins: Membrane and Protein Structural Requirements for Pore Formation.

The cholesterol-dependent cytolysins (CDCs) are a family of bacterial protein toxins specifically targeting eukaryotic cells through the absolute requirement for high concentrations of cholesterol in the target cells' lipid membrane. The soluble monomeric protein secreted by the bacteria oligomerizes on the surface of the target cell, and the complex formed then undergoes a concerted structural transition that results in the creation of a multimeric protein pore. Recognition of the cholesterol-rich membrane by CDCs is a surprisingly subtle process that takes place at the interface between the membrane and surrounding aqueous environment. The structure and composition of the lipid membrane modulates the efficiency with which the protein can identify cholesterol and alters the concentration of sterol required for membrane binding. Some of the details of the interplay between protein and membrane remain to be resolved, and in this review we present a current perspective on CDC pore formation, with particular focus on the role of the lipid bilayer and cholesterol accessibility.

Expression and purification of the native C-amidated antimicrobial peptide maculatin 1.1.

Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from an Australian tree frog and exhibits low micromolar activity against Gram-positive bacteria. The antimicrobial properties of Mac1 are linked to its disruption of bacterial lipid membranes, which has been studied extensively by in vitro nuclear magnetic resonance (NMR) spectroscopy and biophysical approaches. Although in vivo NMR has recently proven effective in probing peptide-lipid interplay in live bacterial cells, direct structural characterisation of AMPs has been prohibited by low sensitivity and overwhelming background noise. To overcome this issue, we report a recombinant expression protocol to produce isotopically enriched Mac1. We utilized a double-fusion construct to alleviate toxicity against the Escherichia coli host and generate the native N-free and C-amidated termini Mac1 peptide. The SUMO and intein tags allowed native N-terminus and C-terminal amidation, respectively, to be achieved in a one-pot reaction. The protocol yielded 0.1 mg/L of native, uniformly 15 N-labelled, Mac1, which possessed identical structure and activity to peptide obtained by solid-phase peptide synthesis.

Water diffusion in complex systems measured by PGSE NMR using chemical shift selective stimulated echo: Elimination of magnetization exchange effects.

The interpretation of molecular translational diffusion as measured by pulsed gradient spin-echo NMR (PGSE NMR) can be complicated by the presence of chemical exchange and/or dipolar cross-relaxation (including relayed cross-relaxation via spin diffusion). The magnitude of influence depends on the kinetics of exchange and/or dipolar cross-relaxation present within the system as well as the PGSE NMR sequences chosen for measurements. First, we present an exchange induced zero-crossing phenomenon for signal attenuation of water in lipidic cubic phases (formed by a mixture of monoolein and water) in the presence of pulsed gradients observed using a standard STimulated Echo (STE) sequence. This magnetization exchange induced zero-crossing phenomenon, a pseudo-negative diffraction-like feature, resembles that reported previously for restricted diffusion when locally anisotropic pores are polydisperse or randomly oriented. We then demonstrate the elimination of these exchange and/or dipolar cross-relaxation induced effects with the use of a chemical shift selective STE (CHESS-STE) sequence, adapted from the previously reported band-selective short transient STE sequence, along with results obtained from the bipolar pulse pair STE sequence for comparison. The CHESS-STE sequence introduced here represents a generic form of PGSE NMR sequences for obtaining water diffusion coefficients free from the influence of exchange and/or dipolar cross-relaxation in complex systems. It has potential applications in measuring translational diffusion of water in biopolymer mixtures as well as probing the microscopic structure in materials via water restricted diffusion measured by PGSE NMR, particularly when the potential presence of exchange/cross-relaxation is of concern.

Nitroxide spin-labeled peptides for DNP-NMR in-cell studies.

Antimicrobial peptides (AMPs) that target lipid membranes show promise as alternatives to conventional antibiotics. However, the molecular mechanisms of membrane perturbation, as most studies are performed in model systems and in-cell structural studies, have yet to be achieved. Solid-state NMR spectroscopy is a valuable technique to investigate peptide-membrane interactions and to determine the structure of peptides, but the short lifespan of bacteria, especially under magic angle spinning conditions, has not permitted in-cell structural studies. Here, we present the first dynamic nuclear polarization (DNP)-NMR in-cell studies of Escherichia coli bacteria incubated with the AMP maculatin 1.1 (Mac1) in combination with novel nitroxide spin-labeled peptides 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC)-[F3W]-Mac1 (MacW) and TOAC-TOAC-MacW. The in-cell 13C and 15N signal NMR enhancements, and 1H spin-lattice T1 relaxation times showed that TOAC-MacW and TOAC-TOAC-MacW performed better than the more hydrophilic biradical AMUPol used for DNP studies. Furthermore, the pores formed by the AMP increased the signal enhancements and decreased T1 values of specifically 13C- and 15N-labeled Mac1. This approach has a great potential for determining the first in situ structures of AMPs in bacteria.-Sani, M.-A., Zhu, S., Hofferek, V., Separovic, F. Nitroxide spin-labeled peptides for DNP-NMR in-cell studies.

Physiochemical Characterization and Stability of Lipidic Cubic Phases by Solution NMR.

Lipidic inverse bicontinuous cubic phases (LCPs), formed via the spontaneous self-assembly of lipids such as monoolein, have found increasing applications in the stabilization and crystallization of integral membrane proteins for structural characterization using X-ray crystallography. Their use as effective drug release matrices has also been demonstrated. Nuclear magnetic resonance (NMR) spectroscopy, both solution and solid state, has previously been employed for the characterization of LCPs and related systems. Herein, we report a number of novel features of solution NMR for probing the fundamental composition and structural properties of monoolein-based LCPs. These include (1) more complete assignments of both 1H and 13C chemical shifts, (2) direct quantification of hydration level in LCPs using one-dimensional (1D) 1H NMR, and (3) monitoring longer-term stability of LCPs and evaluating alterations introduced into standard LCPs at the submolecular level.

Metallo-Cubosomes: Zinc-Functionalized Cubic Nanoparticles for Therapeutic Nucleotide Delivery.

Development of an effective and potent RNA delivery system remains a challenge for the clinical application of RNA therapeutics. Herein, we describe the development of an RNA delivery platform derived from self-assembled bicontinuous cubic lyotropic liquid crystalline phases, functionalized with zinc coordinated lipids. These metallo-cubosomes were prepared from a series of novel lipidic zinc(II)-bis(dipicolylamine) (Zn2BDPA)) complexes admixed with glycerol monooleate (GMO). The zinc metallo-cubosomes showed the high affinity to siRNA through interaction between Zn2BDPA and the phosphate groups of RNA molecules. Using a combination of dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM), we demonstrated that a variety of Zn2BDPA lipid derivatives can be loaded into GMO cubosomes and the introduction of Zn2BDPA lipids effected an internal cubic phase transition of the resulting metallo-cubosomes. The findings of this study lay the foundations for the development of a new class of noncationic lipid-based encapsulation systems, metallo-cubosomes for RNA therapeutic delivery.

In Situ Monitoring of Bacteria under Antimicrobial Stress Using 31P Solid-State NMR.

In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.

Effect of phosphatidylcholine bilayer thickness and molecular order on the binding of the antimicrobial peptide maculatin 1.1.

Antimicrobial peptides (AMPs) interact directly with bacterial membrane lipids. Thus, changes in the lipid composition of bacterial membranes can have profound effects on the activity of AMPs. In order to understand the effect of bilayer thickness and molecular order on the activity of AMPs, the interaction of maculatin 1.1 (Mac1.1) with phosphatidylcholine (PC) model membranes composed of different monounsaturated acyl chain lengths between 14 and 22 carbons was characterised by dual polarisation interferometry (DPI) and 31P and 1H solid-state NMR techniques. The thickness and bilayer order of each PC bilayer showed a linear dependence on the acyl chain length. The binding of Mac1.1 exhibited a biphasic dependency between the amount of bound Mac1.1 and bilayer thickness, whereby the mass of bound peptide increased from C14 to C16 and then decreased from C16 to C22. Significant perturbation of 31P chemical shift anisotropy (CSA) values was only observed for DOPC (C18) and DEPC (C22), respectively. In the case of DEPC, the greater range in CSA indicated different headgroup conformations or environments in the presence of Mac1.1. Overall, the results indicated that there is a significant change in the bilayer order upon binding of Mac1.1 and this change occurred in a co-operative manner at higher concentrations of Mac1.1 with increasing bilayer thickness and order. Overall, an optimum bilayer thickness and lipid order may be required for effective membrane perturbation by Mac1.1 and increasing the bilayer thickness and order may counteract the activity of Mac1.1 and play a role in antimicrobial resistance to AMPs.

Elucidating the bactericidal mechanism of action of the linear antimicrobial tetrapeptide BRBR-NH2.

Linear antimicrobial peptides, with their rapid bactericidal mode of action, are well-suited for development as topical antibacterial drugs. We recently designed a synthetic linear 4-residue peptide, BRBR-NH2, with potent bactericidal activity against Staphylococcus aureus (MIC 6.25 µM), the main causative pathogen of human skin infections with an unknown mechanism of action. Herein, we describe a series of experiments conducted to gain further insights into its mechanism of action involving electron microscopy, artificial membrane dye leakage, solution- and solid-state NMR spectroscopy followed by molecular dynamics simulations. Experimental results point towards a SMART (Soft Membranes Adapt and Respond, also Transiently) mechanism of action, suggesting that the peptide can be developed as a topical antibacterial agent for treating drug-resistant Staphylococcus aureus infections.

Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes.

The Na+,K+-ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na+ and K+ electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state 31P and 2H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations.