Antimicrobial peptides and their superior fluorinated analogues: structure-activity relationships as revealed by NMR spectroscopy and MD calculations

The conformations of two synthetic pentapeptides with antimicrobial activity and their 4-fluorophenylalanine (Pff)-containing analogues (ArXArXAr-NH(2); Ar=Phe, Pff; X=Lys, Arg) have been studied. NMR experiments carried out both in aqueous fluoroalcohol solutions and SDS micelles permitted their interactions with membrane-like environments to be explored. WaterLOGSY experiments and Mn(2+)-based paramagnetic probes were also applied to assess their orientations with respect to the SDS micelles. In addition, pulse-field gradient (PFG) diffusion NMR spectroscopy studies were conducted, under different experimental conditions (i.e., concentration, temperature) to characterize the possible changes in the peptides' aggregation states as a putative critical factor for their antimicrobial activity. Finally, molecular dynamics simulations on a variety of conformations showed the intrinsic flexibility of these peptides in both aqueous solutions and membrane-mimetic systems.     

Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents

For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles. Detailed characterization of the mixed detergent/protein micelles is then of key importance to validate the results from such studies, and to evaluate how faithfully the natural environment of the protein in the biological membrane is mimicked by the micelle. In this paper, a selection of paramagnetic probes with different physicochemical properties are used to characterize the 60 kDa mixed micelles consisting of about 90 molecules of the detergent dihexanoylphosphatidylcholine (DHPC) and one molecule of the Escherichia coli outer-membrane protein X (OmpX), which had previously been extensively studied by solution NMR techniques. The observation of highly selective relaxation effects on the NMR spectra of OmpX and DHPC from a water-soluble relaxation agent and from nitroxide spin labels attached to lipophilic molecules, confirmed data obtained previously with more complex NMR studies of the diamagnetic OmpX/DHPC system, and yielded additional novel insights into the protein-detergent interactions in the mixed micelles. The application of paramagnetic probes to the well-characterized OmpX/DHPC system indicates that such probes should be widely applicable as an efficient support of NMR studies of the topology of mixed membrane protein-detergent micelles.     

Ion and Molecular Transport in Solid Electrolytes Studied by NMR

NMR is the method of choice for molecular and ionic structures and dynamics investigations. The present review is devoted to solvation and mobilities in solid electrolytes, such as ion-exchange membranes and composite materials, based on cesium acid sulfates and phosphates. The applications of high-resolution NMR, solid-state NMR, NMR relaxation, and pulsed field gradient ¹H, ⁷Li, ¹³C, ¹⁹F, ²³Na, ³¹P, and ¹³³Cs NMR techniques are discussed. The main attention is paid to the transport channel morphology, ionic hydration, charge group and mobile ion interaction, and translation ions and solvent mobilities in different spatial scales. Self-diffusion coefficients of protons and Li⁺, Na⁺, and Cs⁺ cations are compared with the ionic conductivity data. The microscopic ionic transfer mechanism is discussed.     

A novel method for determination of polyester end-groups by NMR spectroscopy

An efficient, convenient and quantitative method for characterising polyester end-groups is described. We have found that trichloroacetyl isocyanate (TAI) reacts rapidly and quantitatively with both carboxyl [C(O)OH] and hydroxyl (OH) chain ends to form derivatives that can be readily determined by ¹H NMR spectroscopy. The TAI capped end-groups give rise to characteristic imidic NH resonances in a normally clear region of the ¹H NMR spectrum [δ∼10-11.5 for C(O)-O-C(O)-NH-C(O)CCl₃ from C(O)OH, δ∼8-9 for O-C(O)-NH-C(O)CCl₃ from OH]. The method has been successfully applied to quantitative determination of the end-groups of a wide variety of oligomeric polyesters. It has also been applied to higher molecular weight polyesters including commercial, bottle grade, poly(ethylene terephthalate) (PET) and PET based copolyesters (e.g. PETG).     

Conformation, dynamics, and insertion of a noncysteine-containing protegrin-1 analogue in lipid membranes from solid-state NMR spectroscopy

Disulfide-bonded beta-hairpin structures are common among antimicrobial peptides. Disulfide bonds are known to be important for antimicrobial activity, but the underlying structural reason is not well understood. We have investigated the membrane-bound structure of a disulfide-deleted analogue of the antimicrobial peptide protegrin-1, in which the four Cys residues were replaced by Ala. The secondary structure, dynamics, and topology of this Ala-PG1 peptide in the membrane were determined by using magic-angle-spinning NMR spectroscopy. Conformation-dependent (13)C isotropic chemical shifts of multiple (13)C-labeled residues were obtained from 1D cross-polarization and direct-polarization spectra, and from 2D J-coupling-mediated (13)C-(13)C correlation spectra. Most labeled residues exhibited two conformations: a random coil and a beta-sheet structure. The dual-conformation property was present in both anionic lipid bilayers, which mimic the bacterial membrane, and zwitterionic cholesterol-containing bilayers, which mimic the eukaryotic cell membrane. The mobility of the peptide was measured by using a 2D C-H dipolar-shift correlation experiment. The random-coil fraction was highly mobile whereas the beta-sheet component was rigid. (1)H spin diffusion from the lipid chains to the peptide indicates that the beta-sheet component was well inserted into the anionic membrane, but surface bound in the cholesterol-containing neutral membrane. Thus, the removal of disulfide bonds changed some PG-1 molecules to highly mobile random coils that were poorly associated with the lipid membrane, but other molecules retained a beta-sheet conformation and had a similar membrane-binding topology to the parent peptide. Thus, the reduced antimicrobial activity of Ala-PG1 was largely due to the reduced number of insertion-competent beta-sheet molecules, rather than uniformly weakened activity of identically structured peptides.     

Carbohydrate-protein interactions: a 3D view by NMR

This review focuses on the application of NMR methods for understanding, at the molecular and atomic levels, the diverse mechanisms by which sugar molecules are recognised by the binding sites of lectins, antibodies and enzymes. Given the intrinsic chemical natures of sugars and their flexibility, it is well established that NMR parameters should be complemented by computational methods in attempts to unravel the structural and conformational features of the molecular recognition process unambiguously. We therefore aim here to describe new and significant advances in the knowledge of carbohydrate-protein interactions, obtained by employing state-of-the-art NMR and molecular modelling. We have not attempted to prepare an exhaustive review but have tried to focus on describing the key aspects that should be considered when tackling a problem within this research topic.     

A universal expression tag for structural and functional studies of proteins

Modified ubiquitin sequences, each completed with a His tag and a TEV cleavage site, were designed to enhance the expression of protein/peptide targets. With this new system we have been able to characterize several peptide-protein interactions by ITC and by NMR and CD spectroscopic methods, including the interactions of LIR domains with autophagy modifiers.     

Self-organization Properties of a GPCR-Binding Peptide with a Fluorinated Tail Studied by Fluorine NMR Spectroscopy

Conjugation of the bioactive apelin-17 peptide with a fluorocarbon chain results in self-organization of the peptide into micelles. Fluorine NMR spectroscopy studies show that the fluoropeptide‘s micelles are monodisperse, while proton NMR indicates that the peptide moiety remains largely disordered despite micellization. A very fast exchange rate is measured between the free and micellar states of the peptide which enables the number of molecules present in the micelle to be estimated as 200, in agreement with values found by dynamic light scattering measurements.     

Characterization of Membrane Protein-Lipid Interactions in Unfolded OmpX with Enhanced Time Resolution by Hyperpolarized NMR

Proton nuclear spins of dodecyl phosphocholine molecules below the critical micelle concentration are hyperpolarized by using dissolution dynamic nuclear polarization (D-DNP). NMR signal enhancements of 1210±400 and 1610±550 are obtained at 9.4 T, for choline methyls in the head group of the lipid and for the tail-end methyl group, respectively. This polarization is transferred to the unfolded protein through the nuclear Overhauser effect, after dilution to a final denaturant concentration of 0.8 M urea. As a result, the amide and aromatic side-chain signals of the protein are increased up to sixfold. Selective inversion pulses applied either on the head-group or tail-group of the lipid are used to identify the source of the transferred polarization. The normalized cross-relaxation rates of σ_N,tail=−1.8±0.1 s⁻¹ M⁻¹ and σ_N,head=−0.5±0.3 s⁻¹ M⁻¹ are obtained, showing a larger polarization transfer from the tail groups. These cross-relaxation rates are determined at a low urea concentration, which constitutes refolding conditions for the protein. The sensitivity enhancement by D-DNP permits to access these conditions with a measurement time on the order of seconds, and may further open the possibility to investigate structural changes in membrane proteins during folding.     

Thermal oxidation of model molecules to reveal vegetable oil polymerization studied by NMR spectroscopy and self-diffusion

Oxidative polymerization of plant oils and lipids is poorly understood yet widely encountered. Oils and fats are renewable resources providing biofuels and polymers. Oil oxidation is accelerated at high temperatures, typically above 110°C, where triacylglycerides are converted into toxic compounds and viscous deleterious polymers. Polymerization of mono-unsaturated oil (210°C, 3 h, open to air) was investigated by comparing four similar sized molecules with different functional groups: oleic acid, methyl oleate, trans-7-tetradecene, and stearic acid. Non-volatile products identified by NMR spectroscopy are minor ketones for saturated fatty acid (stearic acid), epoxides for acyl chains without acid groups (methyl oleate, tetradecane) and copious oligomerization, through ester cross-links, for acyl chains with acid, and olefinic groups (oleic acid). Long range C H coupling clearly shows ester (not ether) cross-links, contradicting long-held beliefs. Chain fragmentation also occurs for heated oleic acid as revealed by formation of a species with a methylene group bonded to oxygen of an ester,  CH₂ O C(O) . Large size (slow diffusion) of the first oligomer (trimer) formed from oleic acid, used to represent hydrolyzed vegetable oil, was evidenced by DOSY (diffusion-ordered spectroscopy). Combined NMR results show oligomers found in heated oleic acid are fatty acid estolides. Model oil reactions demonstrate why olefin and carboxylic acid groups are required for polymerization.     

Characterization of siloxane copolymers by solution O NMR spectroscopy

Solution ¹⁷O NMR spectroscopy was used for structure elucidation of siloxane copolymers with the natural abundance of ¹⁷O, i.e. without any enrichment prior to spectroscopy. Homo, co, and terpolymers, as well as linear chains, cyclic oligomers, and graft polymers were investigated. All relevant chemical shifts and corresponding linewidths were reported for siloxane polymers substituted with methyl, phenyl, 3-cyanopropyl, 2-cyanoethyl, 3,3,3-trifluoropropyl, and polyethylene glycol ligands and with the backbone stiffening groups tetramethyl-p-silphenylene, tetramethyl-p,p′-sildiphenylene ether, and m-carborane. An increment system was extended to predict the chemical shifts of substituted siloxane copolymers. ¹⁷O NMR spectroscopy of polysiloxanes provided information concerning their chemical composition, average molecular weight, and microstructure.     

Large-scale in vivo synthesis of the carbohydrate moieties of gangliosides GM1 and GM2 by metabolically engineered Escherichia coli

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc; Gal = galactose, Glc = glucose, Ac = acetyl) and GM1 (Galbeta-3GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc. The GM2 oligosaccharide-producing strain TA02 was devoid of both beta-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP = cytidine monophosphate), alpha-2,3-sialyltransferase, UDP-GlcNAc (UDP = uridine diphosphate) C4 epimerase, and beta-1,4-GalNAc transferase. When this strain was cultivated on glycerol, exogenously added lactose and sialic acid were shown to be actively internalized into the cytoplasm and converted into GM2 oligosaccharide. The in vivo synthesis of GM1 oligosaccharide was achieved by taking a similar approach but using strain TA05, which additionally overexpressed the gene for beta-1,3-galactosyltransferase. In high-cell-density cultures, the production yields for the GM2 and GM1 oligosaccharides were 1.25 g L(-1) and 0.89 g L(-1), respectively.     

Blood Group O→A Transformation by Chemical Ligation of Erythrocytes

Agglutination of red blood cells (RBCs) remains the only practical method for routine use for ABH typing in clinical practice. However, exact mechanistic details of agglutination are not yet thoroughly studied. In this research, RBCs of blood group O were converted to blood group A through two approaches: by chemical ligation of the cells’ glycocalyx with synthetic blood group A tetrasaccharide, and by insertion of synthetic glycolipid carrying the same A antigen into the cells’ membranes. The O→A ligated RBCs and natural A RBCs showed comparable agglutination characteristics with antibodies. As expected, RBCs with inserted glycolipid showed lower agglutination scores. This approach could help cell biologists in site-specific and cell-friendly modification of glycocalyx by other ligands.