Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide.

Microbial polysaccharide characterization requires purification that often involves detergent precipitation and lyophilization. Here we examined physicochemical changes following lyophilization of Cryptococcus neoformans exopolysaccharide (EPS). Solution 1H Nuclear Magnetic Resonance (NMR) reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H solid-state Nuclear Magnetic Resonance (ssNMR) shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide bind native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.

Tailoring NMR experiments for structural characterization of amorphous biological solids: A practical guide.

Many interesting solid-state targets for biological research do not form crystalline structures; these materials include intrinsically disordered proteins, plant biopolymer composites, cell-wall polysaccharides, and soil organic matter. The absence of aligned repeating structural elements and atomic-level rigidity presents hurdles to achieving structural elucidation and obtaining functional insights. We describe strategies for adapting several solid-state NMR methods to determine the molecular structures and compositions of these amorphous biosolids. The main spectroscopic problems in studying amorphous structures by NMR are over/under-sampling of the spin signals and spectral complexity. These problems arise in part because amorphous biosolids typically contain a mix of rigid and mobile domains, making it difficult to select a single experiment or set of acquisition conditions that fairly represents all nuclear spins in a carbon-based organic sample. These issues can be addressed by running hybrid experiments, such as using direct excitation alongside cross polarization-based methods, to develop a more holistic picture of the macromolecular system. In situations of spectral crowding or overlap, the structural elucidation strategy can be further assisted by coupling 13C spins to nuclei such as 15N, filtering out portions of the spectrum, highlighting individual moieties of interest, and adding a second or third spectral dimension to an NMR experiment in order to spread out the resonances and link them pairwise through space or through bonds. We discuss practical aspects and illustrations from the recent literature for 1D experiments that use cross or direct polarization and both homo- and heteronuclear 2D and 3D solid-state NMR experiments.

Solid-state NMR spectroscopy identifies three classes of lipids in Cryptococcus neoformans melanized cell walls and whole fungal cells.

A primary virulence-associated trait of the opportunistic fungal pathogen Cryptococcus neoformans is the production of melanin pigments that are deposited into the cell wall and interfere with the host immune response. Previously, our solid-state NMR studies of isolated melanized cell walls (melanin "ghosts") revealed that the pigments are strongly associated with lipids, but their identities, origins, and potential roles were undetermined. Herein, we exploited spectral editing techniques to identify and quantify the lipid molecules associated with pigments in melanin ghosts. The lipid profiles were remarkably similar in whole C. neoformans cells, grown under either melanizing or nonmelanizing conditions; triglycerides (TGs), sterol esters (SEs), and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and nonmelanized cells, melanin ghosts were relatively enriched in SEs and PPs. In contrast to lipid structures reported during early stages of fungal growth in nutrient-rich media, variants found herein could be linked to nutrient stress, cell aging, and subsequent production of substances that promote chronic fungal infections. The fact that TGs and SEs are the typical cargo of lipid droplets suggests that these organelles could be connected to C. neoformans melanin synthesis. Moreover, the discovery of PPs is intriguing because dolichol is a well-established constituent of human neuromelanin. The presence of these lipid species even in nonmelanized cells suggests that they could be produced constitutively under stress conditions in anticipation of melanin synthesis. These findings demonstrate that C. neoformans lipids are more varied compositionally and functionally than previously recognized.

The Droserasin 1 PSI: A Membrane-Interacting Antimicrobial Peptide from the Carnivorous Plant Drosera capensis.

The Droserasins, aspartic proteases from the carnivorous plant Drosera capensis, contain a 100-residue plant-specific insert (PSI) that is post-translationally cleaved and independently acts as an antimicrobial peptide. PSIs are of interest not only for their inhibition of microbial growth, but also because they modify the size of lipid vesicles and strongly interact with biological membranes. PSIs may therefore be useful for modulating lipid systems in NMR studies of membrane proteins. Here we present the expression and biophysical characterization of the Droserasin 1 PSI (D1 PSI.) This peptide is monomeric in solution and maintains its primarily α -helical secondary structure over a wide range of temperatures and pH values, even under conditions where its three disulfide bonds are reduced. Vesicle fusion assays indicate that the D1 PSI strongly interacts with bacterial and fungal lipids at pH 5 and lower, consistent with the physiological pH of D. capensis mucilage. It binds lipids with a variety of head groups, highlighting its versatility as a potential stabilizer for lipid nanodiscs. Solid-state NMR spectra collected at a field strength of 36 T, using a unique series-connected hybrid magnet, indicate that the peptide is folded and strongly bound to the membrane. Molecular dynamics simulations indicate that the peptide is stable as either a monomer or a dimer in a lipid bilayer. Both the monomer and the dimer allow the passage of water through the membrane, albeit at different rates.

3D-printed dissolvable inserts for efficient and customizable fabrication of NMR transceiver coils.

We describe a simplified method for improving the reproducibility of transceiver coil fabrication for nuclear magnetic resonance (NMR) through single-use templates made from 3D-printed polymer forms. The utility of dissolvable inserts for achieving performance enhanced resonators (DIAPERs) is tested herein by a comparison of RF homogeneity along the rotor axis for variable-pitch solenoids with different inter-turn spacing. Simulated B1 field profiles are compared to experimental homogeneity measurements, demonstrating the potential of this approach for making NMR coils quickly and reproducibly.

Advances in instrumentation and methodology for solid-state NMR of biological assemblies.

Many advances in instrumentation and methodology have furthered the use of solid-state NMR as a technique for determining the structures and studying the dynamics of molecules involved in complex biological assemblies. Solid-state NMR does not require large crystals, has no inherent size limit, and with appropriate isotopic labeling schemes, supports solving one component of a complex assembly at a time. It is complementary to cryo-EM, in that it provides local, atomic-level detail that can be modeled into larger-scale structures. This review focuses on the development of high-field MAS instrumentation and methodology; including probe design, benchmarking strategies, labeling schemes, and experiments that enable the use of quadrupolar nuclei in biomolecular NMR. Current challenges facing solid-state NMR of biological assemblies and new directions in this dynamic research area are also discussed.

Protein structure networks provide insight into active site flexibility in esterase/lipases from the carnivorous plant Drosera capensis.

In plants, esterase/lipases perform transesterification reactions, playing an important role in the synthesis of useful molecules, such as those comprising the waxy coatings of leaf surfaces. Plant genomes and transcriptomes have provided a wealth of data about expression patterns and the circumstances under which these enzymes are upregulated, e.g. pathogen defense and response to drought; however, predicting their functional characteristics from genomic or transcriptome data is challenging due to weak sequence conservation among the diverse members of this group. Although functional sequence blocks mediating enzyme activity have been identified, progress to date has been hampered by the paucity of information on the structural relationships among these regions and how they affect substrate specificity. Here we present methodology for predicting overall protein flexibility and active site flexibility based on molecular modeling and analysis of protein structure networks (PSNs). We define two new types of specialized PSNs: sequence region networks (SRNs) and active site networks (ASNs), which provide parsimonious representations of molecular structure in reference to known features of interest. Our approach, intended as an aid to target selection for poorly characterized enzyme classes, is demonstrated for 26 previously uncharacterized esterase/lipases from the genome of the carnivorous plant Drosera capensis and validated using a case/control design. Analysis of the network relationships among functional blocks and among the chemical moieties making up the catalytic triad reveals potentially functionally significant differences that are not apparent from sequence analysis alone.

Design and construction of a quadruple-resonance MAS NMR probe for investigation of extensively deuterated biomolecules.

Extensive deuteration is frequently used in solid-state NMR studies of biomolecules because it dramatically reduces both homonuclear (1H-1H) and heteronuclear (1H-13C and 1H-15N) dipolar interactions. This approach greatly improves resolution, enables low-power rf decoupling, and facilitates 1H-detected experiments even in rigid solids at moderate MAS rates. However, the resolution enhancement is obtained at some cost due the reduced abundance of protons available for polarization transfer. Although deuterium is a useful spin-1 NMR nucleus, in typical experiments the deuterons are not directly utilized because the available probes are usually triple-tuned to 1H,13C and 15N. Here we describe a 1H/13C/2H/15N MAS ssNMR probe designed for solid-state NMR of extensively deuterated biomolecules. The probe utilizes coaxial coils, with a modified Alderman-Grant resonator for the 1H channel, and a multiply resonant solenoid for 13C/2H/15N. A coaxial tuning-tube design is used for all four channels in order to efficiently utilize the constrained physical space available inside the magnet bore. Isolation among the channels is likewise achieved using short, adjustable transmission line elements. We present benchmarks illustrating the tuning of each channel and isolation among them and the magnetic field profiles at each frequency of interest. Finally, representative NMR data are shown demonstrating the performance of both the detection and decoupling circuits.

Structure prediction and network analysis of chitinases from the Cape sundew, Drosera capensis.

BACKGROUND: Carnivorous plants possess diverse sets of enzymes with novel functionalities applicable to biotechnology, proteomics, and bioanalytical research. Chitinases constitute an important class of such enzymes, with future applications including human-safe antifungal agents and pesticides. Here, we compare chitinases from the genome of the carnivorous plant Drosera capensis to those from related carnivorous plants and model organisms. METHODS: Using comparative modeling, in silico maturation, and molecular dynamics simulation, we produce models of the mature enzymes in aqueous solution. We utilize network analytic techniques to identify similarities and differences in chitinase topology. RESULTS: Here, we report molecular models and functional predictions from protein structure networks for eleven new chitinases from D. capensis, including a novel class IV chitinase with two active domains. This architecture has previously been observed in microorganisms but not in plants. We use a combination of comparative and de novo structure prediction followed by molecular dynamics simulation to produce models of the mature forms of these proteins in aqueous solution. Protein structure network analysis of these and other plant chitinases reveal characteristic features of the two major chitinase families. GENERAL SIGNIFICANCE: This work demonstrates how computational techniques can facilitate quickly moving from raw sequence data to refined structural models and comparative analysis, and to select promising candidates for subsequent biochemical characterization. This capability is increasingly important given the large and growing body of data from high-throughput genome sequencing, which makes experimental characterization of every target impractical.

Sequence comparison, molecular modeling, and network analysis predict structural diversity in cysteine proteases from the Cape sundew, Drosera capensis.

Carnivorous plants represent a so far underexploited reservoir of novel proteases with potentially useful activities. Here we investigate 44 cysteine proteases from the Cape sundew, Drosera capensis, predicted from genomic DNA sequences. D. capensis has a large number of cysteine protease genes; analysis of their sequences reveals homologs of known plant proteases, some of which are predicted to have novel properties. Many functionally significant sequence and structural features are observed, including targeting signals and occluding loops. Several of the proteases contain a new type of granulin domain. Although active site residues are conserved, the sequence identity of these proteases to known proteins is moderate to low; therefore, comparative modeling with all-atom refinement and subsequent atomistic MD-simulation is used to predict their 3D structures. The structure prediction data, as well as analysis of protein structure networks, suggest multifarious variations on the papain-like cysteine protease structural theme. This in silico methodology provides a general framework for investigating a large pool of sequences that are potentially useful for biotechnology applications, enabling informed choices about which proteins to investigate in the laboratory.

Spatial reorientation experiments for NMR of solids and partially oriented liquids.

Motional reorientation experiments are extensions of Magic Angle Spinning (MAS) where the rotor axis is changed in order to average out, reintroduce, or scale anisotropic interactions (e.g. dipolar couplings, quadrupolar interactions or chemical shift anisotropies). This review focuses on Variable Angle Spinning (VAS), Switched Angle Spinning (SAS), and Dynamic Angle Spinning (DAS), all of which involve spinning at two or more different angles sequentially, either in successive experiments or during a multidimensional experiment. In all of these experiments, anisotropic terms in the Hamiltonian are scaled by changing the orientation of the spinning sample relative to the static magnetic field. These experiments vary in experimental complexity and instrumentation requirements. In VAS, many one-dimensional spectra are collected as a function of spinning angle. In SAS, dipolar couplings and/or chemical shift anisotropies are reintroduced by switching the sample between two different angles, often 0° or 90° and the magic angle, yielding a two-dimensional isotropic-anisotropic correlation spectrum. Dynamic Angle Spinning (DAS) is a related experiment that is used to simultaneously average out the first- and second-order quadrupolar interactions, which cannot be accomplished by spinning at any unique rotor angle in physical space. Although motional reorientation experiments generally require specialized instrumentation and data analysis schemes, some are accessible with only minor modification of standard MAS probes. In this review, the mechanics of each type of experiment are described, with representative examples. Current and historical probe and coil designs are discussed from the standpoint of how each one accomplishes the particular objectives of the experiment(s) it was designed to perform. Finally, applications to inorganic materials and liquid crystals, which present very different experimental challenges, are discussed. The review concludes with perspectives on how motional reorientation experiments can be applied to current problems in chemistry, molecular biology, and materials science, given the many advances in high-field NMR magnets, fast spinning, and sample preparation realized in recent years.