Mapping of the Lipid-Binding Regions of the Antifungal Protein NFAP2 by Exploiting Model Membranes.

Fungal infections with high mortality rates represent an increasing health risk. The Neosartorya (Aspergillus) fischeri antifungal protein 2 (NFAP2) is a small, cysteine-rich, cationic protein exhibiting potent anti-Candida activity. As the underlying mechanism, pore formation has been demonstrated; however, molecular level details on its membrane disruption action are lacking. Herein, we addressed the lipid binding of NFAP2 using a combined computational and experimental approach to simple lipid compositions with various surface charge properties. Simulation results revealed binding preferences for negatively charged model membranes, where selectivity is mediated by anionic lipid components enriched at the protein binding site but also assisted by zwitterionic lipid species. Several potential binding routes initiated by various anchoring contacts were observed, which resulted in one main binding mode and a few variants, with NFAP2 residing on the membrane surface. Region 10NCPNNCKHKKG20 of the flexible N-terminal part of the protein showed potency to insert into the lipid bilayer, where the disulfide bond-stabilized short motif 11CPNNC15 could play a key role. In addition, several areas, including the beginning of the N-terminal (residues 1-8), played roles in facilitating initial membrane contacts. Besides, individual roles of residues such as Lys24, Lys32, Lys34, and Trp42 were also revealed by the simulations. Combined data demonstrated that the solution conformation was not perturbed markedly upon membrane interaction, and the folded part of the protein also contributed to stabilizing the bound state. Data also highlighted that the binding of NFAP2 to lipid vesicles is sensitively affected by environmental factors such as ionic strength. Electrostatic interactions driven by anionic lipids were found pivotal, explaining the reduced membrane activity observed under high salt conditions. Experimental data supported the lipid-selective binding mechanisms and pointed to salt-dependent effects, particularly to protein-assisted vesicle aggregation at low ionic strength. Our findings can contribute to the development of NFAP2-based anti-Candida agents and studies aiming at future medical use of peptide-based natural antifungal compounds.

Confirmation of the Disulfide Connectivity and Strategies for Chemical Synthesis of the Four-Disulfide-Bond-Stabilized Aspergillus giganteus Antifungal Protein, AFP.

Emerging fungal infections require new, more efficient antifungal agents and therapies. AFP, a protein from Aspergillus giganteus with four disulfide bonds, is a promising candidate because it selectively inhibits the growth of filamentous fungi. In this work, the reduced form of AFP was prepared using native chemical ligation. The native protein was synthesized via oxidative folding with uniform protection for cysteine thiols. AFP's biological activity depends heavily on the pattern of natural disulfide bonds. Enzymatic digestion and MS analysis provide proof for interlocking disulfide topology (abcdabcd) that was previously assumed. With this knowledge, a semi-orthogonal thiol protection method was designed. By following this strategy, out of a possible 105, only 6 disulfide isomers formed and 1 of them proved to be identical with the native protein. This approach allows the synthesis of analogs for examining structure-activity relationships and, thus, preparing AFP variants with higher antifungal activity.

Dynamic Structures of Bioactive Proteins as Determined by Nuclear Magnetic Resonance.

According to "Panta rhei", a phrase by the ancient Greeks, you cannot enter the same river two times [...].

DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study.

PAF and related antifungal proteins are promising antimicrobial agents. They have highly stable folds around room temperature due to the presence of 3-4 disulfide bonds. However, unfolded states persist and contribute to the thermal equilibrium in aqueous solution, and low-populated states might influence their biological impact. To explore such equilibria during dimethyl sulfoxide (DMSO)-induced chemical unfolding, we studied PAF and its inactive variant PAFD19S using nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). According to the NMR monitoring at 310 K, the folded structures disappear above 80 v/v% DMSO concentration, while the unfolding is completely reversible. Evaluation of a few resolved peaks from viscosity-compensated 15N-1H HSQC spectra of PAF yielded ∆G = 23 ± 7 kJ/M as the average value for NMR unfolding enthalpy. The NMR-based structures of PAF and the mutant in 50 v/v% DMSO/H2O mixtures were more similar in the mixed solvents then they were in water. The 15N NMR relaxation dynamics in the same mixtures verified the rigid backbones of the NMR-visible fractions of the proteins; still, enhanced dynamics around the termini and some loops were observed. DSC monitoring of the Tm melting point showed parabolic dependence on the DMSO molar fraction and suggested that PAF is more stable than the inactive PAFD19S. The DSC experiments were irreversible due to the applied broad temperature range, but still suggestive of the endothermic unfolding of PAF.

The Importance of Mg2+ -Free State in Nucleotide Exchange of Oncogenic K-Ras Mutants.

For efficient targeting of oncogenic K-Ras interaction sites, a mechanistic picture of the Ras-cycle is necessary. Herein, we used NMR relaxation techniques and molecular dynamics simulations to decipher the role of slow dynamics in wild-type and three oncogenic P-loop mutants of K-Ras. Our measurements reveal a dominant two-state conformational exchange on the ms timescale in both GDP- and GTP-bound K-Ras. The identified low-populated higher energy state in GDP-loaded K-Ras has a conformation reminiscent of a nucleotide-bound/Mg2+ -free state characterized by shortened ß2/ß3-strands and a partially released switch-I region preparing K-Ras for the interaction with the incoming nucleotide exchange factor and subsequent reactivation. By providing insight into mutation-specific differences in K-Ras structural dynamics, our systematic analysis improves our understanding of prolonged K-Ras signaling and may aid the development of allosteric inhibitors targeting nucleotide exchange in K-Ras.

Molecular and comparative genomic analyses reveal evolutionarily conserved and unique features of the Schizosaccharomyces japonicus mycelial growth and the underlying genomic changes.

Fungal pathogens, from phytopathogenic fungus to human pathogens, are able to alternate between the yeast-like form and filamentous forms. This morphological transition (dimorphism) is in close connection with their pathogenic lifestyles and with their responses to changing environmental conditions. The mechanisms governing these morphogenetic conversions are still not fully understood. Therefore, we studied the filamentous growth of the less-known, non-pathogenic dimorphic fission yeast, S. japonicus, which belongs to an ancient and early evolved branch of the Ascomycota. Its RNA sequencing revealed that several hundred genes were up- or down-regulated in the hyphae compared to the yeast-phase cells. These genes belonged to different GO categories, confirming that mycelial growth is a rather complex process. The genes of transport- and metabolic processes appeared especially in high numbers among them. High expression of genes involved in glycolysis and ethanol production was found in the hyphae, while other results pointed to the regulatory role of the protein kinase A (PKA) pathway. The homologues of 49 S. japonicus filament-associated genes were found by sequence alignments also in seven distantly related dimorphic and filamentous species. The comparative genomic analyses between S. japonicus and the closely related but non-dimorphic S. pombe shed some light on the differences in their genomes. All these data can contribute to a better understanding of hyphal growth and those genomic rearrangements that underlie it.

Tightly linked morpholino-nucleoside chimeras: new, compact cationic oligonucleotide analogues.

The polyanionic phosphodiester backbone of nucleic acids contributes to high nuclease sensitivity and low cellular uptake and is therefore a major obstacle to the biological application of native oligonucleotides. Backbone modifications, particularly charge alterations is a proven strategy to provide artificial oligonucleotides with improved properties. Here, we describe the synthesis of a new type of oligonucleotide analogues consisting of a morpholino and a ribo- or deoxyribonucleoside in which the 5'-amino group of the nucleoside unit provides the nitrogen of the morpholine ring. The synthetic protocol is compatible with trityl and dimethoxytrityl protecting groups and azido functionality, and was extended to the synthesis of higher oligomers. The chimeras are positively charged in aqueous medium, due to the N-alkylated tertiary amine structure of the morpholino unit.

Solution Structure, Dynamics, and New Antifungal Aspects of the Cysteine-Rich Miniprotein PAFC.

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical ß-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.

Homo- and Heteroassociations Drive Activation of ErbB3.

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.

Synthesis and evaluation of biological activity for dual-acting antibiotics on the basis of azithromycin and glycopeptides.

One of the promising directions of the combined approach is the design of dual-acting antibiotics - heterodimeric structures on the basis of antimicrobial agents of different classes. In this study a novel series of azithromycin-glycopeptide conjugates were designed and synthesized. The structures of the obtained compounds were confirmed using NMR spectroscopy and mass spectrometry data including MS/MS analysis. All novel hybrid antibiotics were found to be either as active as azithromycin and vancomycin against Gram-positive bacterial strains or have superior activity in comparison with their parent antibiotics. One compound, eremomycin-azithromycin conjugate 16, demonstrated moderate activity against Enterococcus faecium and Enterococcus faecalis strains resistant to vancomycin, and equal to vancomycin's activity for the treatment of mice with Staphylococcus aureus sepsis.

Tricyclanos: conformationally constrained nucleoside analogues with a new heterotricycle obtained from a d-ribofuranose unit.

A novel type of nucleoside analogue in which the sugar part is replaced by a new tricycle, 3,7,10-trioxa-11-azatricyclo[5.3.1.05,11]undecane has been prepared by substrate-controlled asymmetric synthesis. 1,5-Dialdehydes obtained from properly protected or unprotected uridine, ribothymidine, cytidine, inosine, adenosine and guanosine by metaperiodate oxidation reacted readily with tris(hydroxymethyl)aminomethane to provide the corresponding tricyclic derivatives with three new stereogenic centers. Through a double cyclisation cascade process the tricyclic compounds were obtained in good to high yields, with very high diastereoselectivity. Formation of one stereoisomer, out of the eight possible, was observed in all cases. The absolute configuration of the new stereotriad-containing tricyclic systems was aided by conventional NMR experiments followed by chemical shift calculations using an X-ray crystal structure as reference that was in good agreement with H-H distances obtained from a new ROESY NMR method. The synthesis was compatible with silyl, trityl and dimethoxytrityl protecting groups. A new reagent mixture containing ZnCl2, Et3SiH and hexafluoroisopropanol was developed for detritylation of the acid-sensitive tricyclano nucleosides.

The dipole potential correlates with lipid raft markers in the plasma membrane of living cells.

The dipole potential generating an electric field much stronger than any other type of membrane potential influences a wide array of phenomena, ranging from passive permeation to voltage-dependent conformational changes of membrane proteins. It is generated by the ordered orientation of lipid carbonyl and membrane-attached water dipole moments. Theoretical considerations and indirect experimental evidence obtained in model membranes suggest that the dipole potential is larger in liquid-ordered domains believed to correspond to lipid rafts in cell membranes. Using three different dipole potential-sensitive fluorophores and four different labeling approaches of raft and nonraft domains, we showed that the dipole potential is indeed stronger in lipid rafts than in the rest of the membrane. The magnitude of this difference is similar to that observed between the dipole potential in control and sphingolipid-enriched cells characteristic of Gaucher's disease. The results established that the heterogeneity of the dipole potential in living cell membranes is correlated with lipid rafts and imply that alterations in the lipid composition of the cell membrane in human diseases can lead to substantial changes in the dipole potential.

rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry.

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.

BACKGROUND: Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. RESULTS: The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays. CONCLUSION: This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure-function analyses.

Offset-compensated and zero-quantum suppressed ROESY provides accurate 1 H-1 H distances in small to medium-sized molecules.

A robust version of the off-resonance ROESY pulse scheme is suggested for the measurement of proton-proton distances or slow chemical exchange in small to medium-sized molecules. The method implements adiabatic ramps to establish a pair of opposite frequency off-resonance spin lock fields - with optionally randomized duration - and adiabatic inversion pulses with simultaneous gradients for efficient zero-quantum suppression. The amended pulse sequence yields pure absorption cross-peaks and works safely for small to medium-sized molecules. The applicability of the method has been demonstrated using small, rigid molecules (strychnine and codeine) and was also applied for a cyclic peptide and a small protein. We found that the pure phase cross-peaks of the new ROESY version are beneficial for distance measurements. The one-dimensional (selective) version of the new method is also powerful for measuring selected pair-wise interactions and distance determination. Copyright © 2016 John Wiley & Sons, Ltd.

Real-time pure shift ¹5N HSQC of proteins: a real improvement in resolution and sensitivity.

Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t 2) broadband homodecoupling, suppressing the multiplet structure caused by proton-proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to (15)N, while the former selects a region of the (1)H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.

"Invisible" conformers of an antifungal disulfide protein revealed by constrained cold and heat unfolding, CEST-NMR experiments, and molecular dynamics calculations.

Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20-40 % at 298 K in a disulfide-rich protein. In addition, sensitive (15) N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR "dark matter". Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction.

Maximum likelihood estimation of FRET efficiency and its implications for distortions in pixelwise calculation of FRET in microscopy.

Ratiometric determination of the efficiency of fluorescence or Förster resonance energy transfer (FRET) is one of the most widespread methods for the characterization of protein clustering and conformation. Low photon numbers, often present in pixel-by-pixel determination of FRET efficiency in digital microscopy, result in large uncertainties in the derived FRET parameter. Here, we propose a method based on maximum likelihood estimation (MLE) of FRET efficiency using photon counting detectors to overcome this limitation. Intensities measured in the donor, FRET, and acceptor channels were all assumed to follow Poisson statistics as a result of detector shot noise. The joint probability of photon numbers detected in the donor, FRET, and acceptor channels was derived using an equation describing the relationship between the three measured intensities. The FRET efficiency generating the measured photon numbers with the largest likelihood was determined iteratively providing a single FRET value for all pixels in the calculation. Since as few as 100 pixels are sufficient to provide a maximum likelihood estimate for FRET, biological variability in FRET values can be revealed by performing the analysis for regions of interests in an image. Since the algorithm provides the probability of a combination of donor, FRET, and acceptor intensities observed in each individual pixel given a certain FRET efficiency, outlier pixels with low probabilities could be excluded from the analysis. Simulations carried out with low photon numbers in the presence and absence of outlier pixels revealed that the proposed approach can reliably and reproducibly estimate FRET efficiency. In addition, systematic evaluation of the simulation results showed that the distribution of pixel-by-pixel FRET efficiencies is skewed, and the mean of these FRET values is a biased and unreliable estimate of the FRET efficiency. In the absence of outlier pixels, FRET calculated from summed donor, FRET, and acceptor intensities proved to be as reliable as MLE. We conclude that MLE of FRET outperforms calculations using summed and pixel-by-pixel intensities in biologically relevant situations involving low photon numbers and outlier pixels. © 2014 International Society for Advancement of Cytometry.

Production of a defensin-like antifungal protein NFAP from Neosartorya fischeri in Pichia pastoris and its antifungal activity against filamentous fungal isolates from human infections.

Neosartorya fischeri NRRL 181 isolate secretes a defensin-like antifungal protein (NFAP) which has a remarkable antifungal effect against ascomycetous filamentous fungi. This protein is a promising antifungal agent of biotechnological value; however in spite of the available knowledge of the nature of its 5'-upstream transcriptional regulation elements, the bulk production of NFAP has not been resolved yet. In this study we carried out its heterologous expression in the yeast Pichia pastoris and investigated the growth inhibition effect exerted by the heterologous NFAP (hNFAP) on filamentous fungal isolates from human infections compared with what was caused by the native NFAP. P. pastoris KM71H transformant strain harboring the pPICZαA plasmid with the mature NFAP encoding gene produced the protein. The final yield of the hNFAP was sixfold compared to the NFAP produced by N. fischeri NRRL 181. Based on the signal dispersion of the amide region, it was proven that the hNFAP exists in folded state. The purified hNFAP effectively inhibited the growth of fungal isolates belonging to the Aspergillus and to the Fusarium genus, but all investigated zygomycetous strain proved to be insusceptible. There was no significant difference between the growth inhibition effect exerted by the native and the heterologous NFAP. These data indicated that P. pastoris KM71H can produce the NFAP in an antifungally active folded state. Our results provide a base for further research, e.g., investigation the connection between the protein structure and the antifungal activity using site directed mutagenesis.

Synthesis of a cluster-forming sialylthio-D-galactose fullerene conjugate and evaluation of its interaction with influenza virus hemagglutinin and neuraminidase.

In order to obtain self assembling, multivalent ligand for influenza virus hemagglutinin α-N-acetylneuraminyl-(2-6)-D-galactopyranose has been synthesized and bonded to a water soluble fullerene derivative using 1,3-dipolar cycloaddition click reaction. The aggregating amphiphilic compound did not inhibit the influenza virus hemagglutinin, but it proved to be an inhibitor of its neuraminidase with a 50% inhibitory concentration of 81 µM.