PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2015 May 29; 290(22): 13779–13790.
Published online 2015 March 30. doi:  10.1074/jbc.M114.618389
PMCID: PMC4447955

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall*

Abstract

Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D 13C-13C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics.

Keywords: biomaterials, biophysics, fungi, melanogenesis, solid state NMR, structural biology, Cryptococcus neoformans, eumelanin, fungal melanin

Introduction

Among the natural pigments that are used increasingly to guide the design of therapeutic and “smart” energy conversion materials (1,3), black or brown fungal eumelanins have attracted particular interest because of their versatile roles as virulence factors, in drug resistance, and in protection from UV radiation (4, 5). Nonetheless, elucidating the molecular-scale basis for these important properties has been challenging because the materials are insoluble, heterogeneous, and amorphous in structure. Despite spectroscopic and structural reports on melanins from diverse biological sources (2, 3, 6,10), the detailed molecular architecture of these natural pigments within their cellular milieu has remained unresolved.

The pathogenic Cryptococcus neoformans fungus has provided a unique investigative system for melanin biopolymer structure because this organism uses obligatory exogenous catecholamine precursors to produce the natural pigment. Hence, in contrast to other sources of natural melanins (6, 7, 9, 10), the starting materials and corresponding metabolic products for C. neoformans melanization can be well defined. Furthermore, we can selectively isolate for investigation those cellular constituents that are closely associated with the pigment and thereby protected from both environmental effects and chemical degradation. Finally, high resolution solid-state nuclear magnetic resonance (NMR) approaches can yield direct insights into the atomic level structure, dynamics, and action mechanisms of noncrystalline bioassemblies, including plant and microbial complexes that have polysaccharide or lipid constituents (11,16). Thus, it is feasible to circumvent the difficulties of solubilization or crystallization to access macromolecular structure for pigments derived from known small molecule isotopically enriched precursors by using the C. neoformans fungal melanin and solid-state NMR.

For instance, we have produced C. neoformans “ghosts” consisting exclusively of melanin and the cell-wall remnants from melanized fungal cells (17, 18), which enable us to monitor the metabolic fate of both l-dopa and mannose or glucose “feedstocks,” to track the molecular development of precursors containing 13C isotopic labels at defined molecular sites, and to test mechanistic hypotheses for C. neoformans melanin biosynthesis by systematically varying the catecholamine precursors (8, 19, 20). To date, the eumelanin structural arrangements for indole-based aromatics, cell wall-derived polysaccharide components, and associated “lipid-like” aliphatic moieties have been deduced partially and indirectly from chemical shift trends observed in cross-polarization magic-angle spinning (CPMAS)3 NMR experiments on intact solid samples or bonded spin-spin interactions observed by high resolution MAS of the aliphatic fraction of the pigment-cell wall assembly that is capable of swelling (8, 19,21). Although the major 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid building blocks have been deduced by chemical analysis of degraded melanins (1, 22, 23), key architectural questions regarding the aromatic core of the intact pigment produced in cell-free or fungal systems have just begun to be addressed (24). The pigment associations and covalent connections to the cell-wall constituents remain uncertain, despite their essential functional roles for C. neoformans melanin cellular protection, treatment of infections, and energy trapping (4, 5, 25).

A major unsolved question in fungal cell biology focuses on the process by which melanin is incorporated into cell walls to generate structures that enhance structural hardiness and diminish susceptibility to immune defense mechanisms. For eumelanin embedded in the innermost layer of polysaccharide cell walls of the C. neoformans fungus and proximal to the phospholipid cell membrane (26, 27), this study focuses on analyzing the structural framework of the pigmented assemblies formed from obligatory catecholamine and glucose starting materials and then isolated from the fungal cells as chemically resistant melanin ghosts. A suite of 1- and 2D 13C solid-state NMR experiments was used to address several important open structural questions as follows. 1) What are the developmental time frames that characterize the formation of indole-based aromatics, oxygenated carbons from cell-wall polysaccharides, and fatty acyl-based cell membrane constituents within the melanizing fungal cells? 2) What kinds of polysaccharide and acylglyceride molecular frameworks, or co-organized scaffolds, are formed by C. neoformans glucose metabolism in the presence of l-dopa? 3) Which indole-derived moieties from the obligatory l-dopa precursor are spatially close and/or covalently linked to the glucose-derived constituents of the cell wall in melanized C. neoformans cells? 4) Which side chain-derived functional moieties from the obligatory l-dopa precursor are spatially close and/or covalently connected to the glucose-derived constituents of the cell wall in melanized C. neoformans cells?

Hence, in addition to probing the (supra)molecular architectures of the cell-wall polysaccharides and indole-based polymers individually at atomic scale, we have examined their intercomponent spatial proximities and covalent linkages within the melanin assembly of C. neoformans. Given the ubiquity of melanin pigments in all biological kingdoms, our fundamental studies can have important practical consequences for medical therapeutics, environmental remediation agents, protective coatings, and drug carriers (2,4).

Experimental Procedures

Melanin Biosynthesis in C. neoformans

The serotype D 24067 strain of the C. neoformans fungus (American Type Culture Collection 208821) was incubated with 1 mm solutions of l-dopa or dopamine substrates in chemically defined media (29.4 mm KH2PO4, 10 mm MgSO4, 13 mm glycine, 15 mm d-glucose, and 3 μm thiamine, all from Sigma), as described previously (17, 18, 28); in designated experiments, these materials were supplied as l-[ring-U-13C6]dopa, l-[2,3-13C2]dopa, and/or [U-13C6]glucose (from Cambridge Isotope Labs, Andover, MA). The cells were grown at 30 °C for periods of 4–14 days in separate experiments, using a rotatory shaker operating at 150 rpm.

Fungal cell pellets were obtained by centrifugation at 2000 rpm and washed with phosphate-buffered saline (PBS) to isolate melanin ghosts for biophysical study. Cell walls were removed by suspending the cells in 1.0 m sorbitol, 0.1 m sodium citrate, pH 5.5, and incubating for 24 h at 30 °C with 10 mg/ml lysing enzymes from Trichoderma harzianum. Centrifugation at 2000 rpm for 10 min yielded a pellet of melanized protoplasts that was washed several times with PBS to obtain a nearly clear supernatant. To denature proteinaceous materials, the melanized cell suspension was incubated with 4 m guanidine thiocyanate for 12 h at room temperature in a rocker (Shaker 35, Labnet, Woodbridge, NJ). The recovered cell debris was collected, washed 2–3 times with ~20 ml of PBS, and then incubated for 4 h at 65 °C in 5 ml of buffer (10 mm Tris-HCl, pH 8.0, 5 mm CaCl2, 5% SDS) containing 1 mg/ml proteinase K (Roche Applied Science). The cell debris was recovered, washed 2–3 times with ~20 ml of PBS, and then subjected to three successive Folch lipid extractions (29) while maintaining the proportions of chloroform, methanol, and saline solution in the final mixture as 8:4:3. To hydrolyze cellular contaminants associated with melanin, the final product was suspended in 20 ml of 6 m HCl and boiled for 1 h. The black particles that survived HCl treatment retain the cellular shape of melanized C. neoformans cells and are known as melanin ghosts; they correspond to melanin pigments and pigment-bound cellular components. These particles were dialyzed against distilled water for 14 days with daily water changes and then lyophilized. The reproducibility of the protocol was tested by repeating the extraction process with two different batches of C. neoformans pigments produced with each of the catecholamine precursors.

Solid-state NMR

Solid-state NMR measurements were carried out using either of two instruments as follows: a Varian (Agilent) DirectDrive 1 NMR spectrometer operating at a 1H frequency of 600 MHz and equipped with a 1.6-mm HXY fastMAS probe filled with 2–6 mg of powdered sample and spinning typically at 15 kHz (±20 Hz) (Agilent Technologies, Santa Clara, CA); or a Bruker Avance I spectrometer (Bruker BioSpin Corp., Billerica, MA) operating at a 1H frequency of 750 MHz and equipped with 4-mm HX, 3.2-mm HCN, or 3.2 mm HCN E-free probes containing 6–18 mg of powdered sample and spinning typically at 15 kHz (±5 Hz). All spectra were acquired at spectrometer-set temperatures of 25 °C.

Typical 90° pulse lengths for 1H were ~2.5 μs for the Bruker HCN probe and ~3 μs for the HCN E-free probe; 13C 90° pulse lengths were ~5 μs for both the 4-mm HX and 3.2-mm HCN probes. For the 1.6-mm Varian HXY fastMAS probe, typical 90° pulse lengths were ~1.2 μs for 1H and ~1.3 μs for 13C. The 1D 13C spectral datasets were processed with 50–200 Hz of line broadening; chemical shifts were referenced externally to the methylene (–CH2–) group of adamantane (Sigma) at δC = 38.48 ppm (30).

For 1D 13C NMR using the 3.2-mm Bruker probes, ~20–50% linearly ramped radiofrequency (rf) field strengths (31) for 1H and a 50 kHz constant rf field for 13C were applied with typical 1–3-ms CP times to transfer magnetization from 1H to 13C nuclear spin baths; 80–100 kHz high power heteronuclear proton decoupling was applied using the two-pulse phase-modulated pulse sequence (32, 33); 3-s recycle delays were inserted between successive scans. Typical experimental parameters on the Bruker spectrometer included ~100 kHz sweep width, ~10–15-ms acquisition time, and 128–1024 transients for 13C-enriched samples.

For low yield samples (<5 mg), 1D 13C CPMAS spectra were recorded with the 1.6-mm Varian probe using typical 1–3-ms cross-polarization times with ramped field strengths as described above. High power heteronuclear 1H decoupling (175–185 kHz) was achieved using the small phase incremental alternation pulse sequence (33), and acquisition was carried out with a 3-s recycle delay. Typical experimental parameters (16, 20, 24) on the Varian spectrometer included 46 kHz sweep width, ~25-ms acquisition time, and 128–1024 transients for 13C-enriched pigments. 13C multiple CP experiments (34) were validated against traditional direct polarization measurements and used to obtain high throughput quantification of the pigment composition. The recycle delays at the beginning of the multiple CP experiments were 3 s, and the duration of the repolarization period was 0.8 s for the natural abundance pigments. Cross-polarization times of 1.0 ms with 10 recursive cycles were used for the multiple CP measurement, and 5000–6000 transients were acquired for natural abundance samples.

The 2D 13C-13C through-space correlation spectra were collected on 13C-enriched melanin samples using radiofrequency field-assisted diffusion mixing implemented in a dipolar assisted rotational resonance (DARR) mixing experiment (35, 36) with the Bruker HCN and HX probes or the Varian HXY probe. The 2D 13C-13C correlation spectra were collected with 25–500-ms mixing times, typical MAS rates of 15 kHz, and 80–100 kHz two-pulse phase-modulated 1H decoupling during acquisition in separate experiments. The small phase incremental alternation pulse sequence was implemented to achieve 175–185 kHz 1H decoupling during acquisition with the Varian HXY probe. 1H-13C cross-polarization was accomplished with a 13C field of ~50 kHz and a proton field strength that was ramped up to 90 kHz during 1–3-ms mixing times. Proton irradiation with a field strength corresponding to 15 kHz was applied during the DARR mixing period for both uniformly and selectively 13C-enriched samples. Spectral widths of ~46–100 kHz in each 13C dimension were used in separate experiments, defined by 1024–2048 points in the direct dimension, 128–1024 scans (direct dimension), and 96–360 points in the indirect dimension. The time proportional phase incrementation method (37) or the small phase incremental alternation pulse sequence (33) were utilized for phase-sensitive detection of the 2D spectra. For some C. neoformans melanin pigments, two or three identical data sets were added together to produce final DARR spectra with increased sensitivity.

Through-bond 13C-13C interactions were measured for 13C-enriched melanins with 2D sensitive absorptive refocused scalar correlation spectroscopy (SAR-COSY) experiments (38) using the Bruker spectrometer operating at a 1H frequency of 750 MHz. The samples were spun in a 3.2-mm HCN E-free probe with 15 kHz MAS, and ~80–90 kHz of two-pulse phase-modulated heteronuclear decoupling was applied. Typical delays for refocusing (4 ms), z-filtering (6 ms), and spin lattice relaxation (3 s) were used in these experiments. Spectral widths of ~100 kHz were used in both direct and indirect dimensions, defined by 890–2048 and 40–136 points, respectively, and 512–1024 scans (direct dimension). For some C. neoformans samples, two identical data sets were added together to produce the final SAR-COSY spectra. Pure phase 2D line shapes were obtained using the States time proportional phase incrementation method (37). For 2D 13C-13C spectral data, an exponential apodization function with 100–200 Hz line broadening was used for both direct and indirect detected dimensions. Identical 1D 13C spectra were recorded before and after lengthy 2D NMR experiments to confirm sample stability.

Results

Time Course of C. neoformans Melanin Development

Solid-state NMR spectra of the melanin ghosts were examined at two stages of cellular melanization, 4 and 14 days after the start of cell growth, to probe the temporal progression of molecular events involved in C. neoformans melanin biosynthesis. As noted above, our isolation treatments ensured that the spectroscopic characterization pertained exclusively to those cellular components that are bound to the pigment. These temporal comparisons included eumelanins derived from laccase-catalyzed polymerization of both l-dopa and dopamine exogenous precursors.

Fig. 1 illustrates a structural comparison between the quantitatively reliable 1D 13C multiple cross-polarization magic-angle spinning (multi-CPMAS) spectra of natural abundance l-dopa C. neoformans melanins at these two growth stages (Fig. 1); aliphatic frameworks displaying similar resonances were present at both early and late times, but the typically broad envelope of prominent aromatic signals does not appear prominently in the solid-state 13C NMR spectrum until later in metabolic development. This result indicates that changes in the melanin structure with time include progressive aromatization of the pigment in the cell wall.

FIGURE 1.
Left, 1D solid-state multi-CPMAS 13C NMR spectra of natural abundance C. neoformans (CN) strain 24067 l-dopa melanins obtained at a 1H frequency of 600 MHz and 15 kHz MAS. Top, mature melanin ghosts isolated after 14 days of cellular biosynthesis. Bottom, ...

From the perspective of molecular structural units, the 4-day 13C NMR spectrum exhibits long-chain methylenes (20–35 ppm), oxymethylene and oxymethine groups from the polysaccharide cell wall (60–105 ppm), alkenes (126–130 ppm), and carboxylate or amide groups (~170–172 ppm) (Table 1). Because all unbound cell membrane polysaccharides, lipids, and protein constituents are removed by the exhaustive treatments used during isolation of the melanin ghosts, the surviving cell wall and cell membrane materials contributing to the spectra of Fig. 1 are taken to be functionalized by the developing pigment moieties to withstand the extractive, enzymatic, and acid chemical procedures. At 14 days, these aliphatic resonances were retained (indicating structural similarities to the 4-day time point) and broadened (suggesting superposition of spectral features from structurally similar cell-wall constituents). The corresponding 13C NMR spectrum at the latter time includes significant contributions from a broad aromatic spectral envelope (110–160 ppm), including protonated and nonprotonated carbons distinguished by dipolar dephasing for C. neoformans melanins made with l-dopa and methyl-l-dopa precursors (8, 19, 20, 24).

TABLE 1
13C NMR resonance assignments for C. neoformans melanins

We observed an analogous developmental trend in multi-CPMAS 13C NMR spectra of C. neoformans dopamine melanins (Fig. 1); the broad aromatic spectral contributions were more prominent with respect to the aliphatics after 14 days compared with 4 days of cell culture. The significant aromatic-to-carboxylate ratios measured herein for both mature pigments also rule out residual protein contributions to the 13C NMR spectra (20). Notably, these comparisons of C. neoformans ghost spectra derived from melanization of the two catecholamine precursors in parallel experiments reveal more rapid development of the aromatic constituents (110–160 ppm) with respect to carboxylate or amide groups (~170–172 ppm) for the dopamine melanins as a function of time.

The developmental studies with l-dopa and dopamine precursors each demonstrate that the assembly of a chemically resistant aliphatic molecular framework takes place prior to significant deposition of acid-resistant oligomeric or polymeric aromatic pigments in the melanized fungal cells, indicating that the aliphatic moieties could form a supporting scaffold for the biosynthesized eumelanins.

Aliphatic Molecular Scaffolds for C. neoformans Melanin Pigment Deposition

The discovery of early developing insoluble aliphatic constituents, which could nonetheless survive the degradative treatments used to isolate fungal melanin ghosts, prompted a detailed spectroscopic examination of the carbon-based molecular frameworks formed in two C. neoformans cell lines. Once again, we isolated only cellular components that were bound to the melanin pigment. A d-[U-13C6]glucose sugar source along with a natural abundance l-dopa catecholamine precursor were first used to focus on the carbon skeleton(s) involving the alkyl, alkoxy, alkene, carboxylate, and amide groups (Fig. 2). For mature 14-day l-dopa pigments in C. neoformans prepared in d-[U-13C6]glucose, our 2D 13C-13C DARR (35, 36) and SAR-COSY (38) measurements (Fig. 3) resolved many overlapping NMR signals and identified both through-space and through-bond pairwise spin connectivities, respectively, in the intact solid melanin ghosts. These 13C-13C interactions established molecular-level structural constraints for 13C nuclei of the melanized polysaccharide cell walls and/or cell membranes, either within a single molecular species or between pairs of constituents within a defined architectural composite.

FIGURE 2.
1D Solid-state spectra for C. neoformans (CN) melanins produced in the 24067 C. neoformans fungal cell line. Left, 1D CPMAS 13C NMR spectrum obtained for C. neoformans melanin produced with natural-abundance l-dopa and d-[U-13C6]glucose, at a 1H frequency ...
FIGURE 3.
Solid-state NMR results, obtained at a 1H frequency of 750 MHz and 15 kHz MAS, for C. neoformans melanins produced with natural-abundance l-dopa and d-[U-13C6]glucose in the 24067 fungal cell line. Left to right, 2D contour plots from DARR experiments ...

Many of the resonances observed in the 2D NMR experiments were in accord with reported spectra for the major cell-wall glucan, chitin, mannan, mannoprotein, and phospholipid constituents (39,43), allowing us to make structural assignments and propose molecular building blocks corresponding to the major C. neoformans melanin resonances of the 13C-enriched constituents (Table 1). For instance, solid-state 13C NMR spectra of insect chitins similar to the N-acetylglucosamine-based polysaccharides implicated in fungal melanization (44, 45) have been assigned to 56–105 ppm (for the ring carbons) and to 24 and 172 ppm (for the acetamido side chain) (39, 43). Through-space and through-bond interactions that support our assignments are detailed below.

We also observed chemical shift discrepancies, which were attributed to cell-wall structural alterations during melanization (4) and an isolation protocol selecting pigment-bound aliphatic constituents that could withstand exhaustive chemical treatments (17, 28). The network of through-space cross-correlated carbon pairs became more extended, as expected, with increasing DARR mixing time from 50 to 250 to 500 ms (Fig. 3). Cross-peaks observed with a 50-ms DARR mixing time reflect primarily short carbon-carbon distances (e.g. 171 × 22 ppm within a chitin N-acetylglucosamine unit) (39, 43). By contrast, data from the 250- and 500-ms mixing periods displayed additional cross-peaks corresponding to pairwise 13C-13C distances as long as ~6 Å (46) (e.g. 126 × 31 ppm between alkene and chain methylene groups and 171 × 72 ppm between carboxylates/amides and oxymethines). These latter aliphatic proximal 13C pairs could arise from acylglycerides, glycosides, or distinct molecular precursors that combine to form a new covalently bonded structure. Notably, our long mixing time DARR spectra include carbon pairs attributable to different C. neoformans aliphatic cell-wall constituents found in closely associated but nonbonded arrangements (45) within the melanin ghost assembly, e.g. carbons derived from β-glucans paired with chitin acetamido methyl groups ((74 × 22), (82 × 22), and (104 × 22) ppm), glyceride esters, or chitin acetamido carbonyl paired with β-glucan and alkene moieties ((171 × 101), (171 × 82), and (171 × 126) ppm).

Taken together, the current analyses of intact solid samples using higher magnetic field strengths and 2D spectroscopy yield a multicarbon structural network of chemically resistant aliphatic moieties resulting from glucose metabolism. Our through-space connectivity evidence for both glycoside and acyl chain structures in l-dopa C. neoformans melanin ghosts augments prior through-bond structural information deduced from 1D 13C CPMAS and solvent-mediated 1H high resolution MAS experiments (19). Moreover, unlike prior efforts to identify bonded 1H-13C chemical fragments within single molecular constituents that are capable of swelling, 2D NMR experiments on solid melanin ghost samples can directly reveal interactions of spatially close pairs of different polysaccharide- and/or acylglyceride-based structural moieties. Figs. 113 show the formation of chemically resistant fungal cell-wall molecular architectures after 4 days of incubation with catecholamine precursors, composed of multiple proximal aliphatic molecular precursors and thus providing a scaffolding support upon which melanin pigments can be deposited as development proceeds. As illustrated under “Spatial Proximity and Covalent Bonding of C. neoformans Melanin Molecular Structures Derived from Glucose and l-Dopa Benzenoid Moieties” and “Spatial Proximity and Covalent Bonding of C. neoformans Melanin Moieties Derived from Glucose and l-Dopa Side Chain” below, the DARR cross-peak signature obtained for d-[U-13C6]glucose-enriched l-dopa melanin also provides essential reference data for the interpretation of analogous 2D spectra of melanized C. neoformans “dual-enriched” with d-[U-13C6]glucose and either l-[2,3-13C2]dopa or l-[ring-13C6]dopa precursors.

The majority of the nearest proximal carbon pairs in the DARR spectra were also confirmed to be one-bond covalent connections using SAR-COSY data (Fig. 3) (47). Cross-peaks were evident for structural fragments in glucose-derived cellular components that were likely to span a broad range of mobilities in this amorphous material. For instance, these pairs were observed within rigid pyranose sugar rings (101 × 52 ppm and 82 × 72 ppm) and for the acetamido group of chitin (171 × 22 ppm). Additional through-bond cross-peaks of a 169 ppm carboxylate resonance with a –CHn at 56 and 52 ppm could correspond to methoxy groups and/or chemically derivatized glucans such as N-acetylglucosamine in chitin (39). The alkenes at 126 ppm could be linked with mobile chain methylene carbon nuclei resonating at 31 ppm, in accord with our prior 2D high resolution MAS NMR observation of through-bond acyl chain signatures in solvent-swelled fungal melanins (19) and the phosphatidylcholine structures that, along with ergosterol, are expected to dominate fungal membranes (27).

Spatial Proximity and Covalent Bonding of C. neoformans Melanin Molecular Structures Derived from Glucose and l-Dopa Benzenoid Moieties

To investigate the macromolecular architecture of the fungal melanin assembly, we began by comparing DARR experiments that displayed through-space 13C-13C connectivities for melanized ghosts with three patterns of isotope enrichment as follows: (a) d-[U-13C6]glucose and l-[12C]dopa (described under “Aliphatic Molecular Scaffolds for C. neoformans Melanin Pigment Deposition”); (b) d-[12C]glucose and l-[ring-13C6]dopa; and (c) dual-labeled d-[U-13C6]glucose and l-[ring-13C6]dopa. As shown in the spectra and contour diagrams of Figs. 3 and and4,4, the purple spectra of sample a are dominated by 13C-enriched aliphatic and carboxyl moieties, whereas the blue spectra of sample b are dominated by 13C-enriched indole-based aromatic resonances, and the red spectra of sample c display contributions from both isotopically enriched constituents. Provisional chemical shift assignments are again summarized in Table 1.

FIGURE 4.
Solid-state NMR at 15 kHz MAS and 1H frequencies of 600 or 750 MHz for C. neoformans melanins produced in the 24067 fungal cell line with l-[ring-U-13C6]dopa and either [U-13C6]glucose (red traces) or [12C]glucose (blue traces) in separate experiments. ...

By accounting for the aliphatic cross-peak proximities identified in the DARR spectra of aliphatic-enriched sample a (Fig. 3), as well as the DARR cross-peaks shown in Fig. 4 for the l-[ring-13C6]dopa melanin sample b, we can identify the functional groups in the dual-labeled sample c that exhibit proximal intercomponent spatial arrangements. Comparable cross-peak features and spectral resolution are observed at 600- and 750-MHz operating frequencies, supporting the overlap of resonances from structurally similar carbons as a prime contributor to the NMR linewidths.

Single-component cross-peaks are attributed to 13C-13C proximities within or between indole rings (no glucose enrichment, blue) and to aliphatic constituents derived from d-[U-13C6]glucose (purple; glucose-only enrichment, Fig. 3). The cross-peaks corresponding to the two-component interactions, which are highlighted with black circles in Fig. 4, position particular indole ring carbons (130 and 144 ppm) within ~6 Å of long-chain methylenes (26 ppm). We would expect to observe an increasing number of aromatic-aliphatic cross-peaks as the DARR mixing time increases from 50 to 500 ms, and the “reach” of the experiment is lengthened, but there is a notable paucity of such features even at the longest mixing time. This latter observation indicates that most pigment rings are situated close to other pigment rings, supporting either lateral aromatic-aromatic interactions or the layered stacking of pigment units deduced using computational and spectroscopic approaches (48,52).

The corresponding SAR-COSY plot identifies one-bond 13C-13C connections, confirming bonding patterns for the polysaccharide- and acyl chain-containing constituents shown in Fig. 3 for the d-[U-13C6]glucose sample and validating linkages within the rigid benzenoid ring. As the SAR-COSY plot displays no cross-peaks with chemical shifts corresponding to the intercomponent through-space 13C-13C interactions, we can rule out direct covalent bonding between the l-[ring-13C6]dopa- and d-[U-13C6]glucose-derived 13C-enriched sites in this C. neoformans melanin sample. Thus, these three sets of 2D NMR data support an architecture in which the benzenoid aromatic ring of the melanin pigment can be associated hydrophobically with the acylglyceride chains of the aliphatic scaffold.

Spatial Proximity and Covalent Bonding of C. neoformans Melanin Moieties Derived from Glucose and l-Dopa Side Chain

The strategy used to deduce relative spatial relationships of benzenoid and aliphatic moieties in the fungal melanin ghosts was adapted to evaluate the proximity and covalent bonding patterns involving macromolecular structures originating from d-glucose and primarily pyrrole (8, 20, 24) pigment moieties that were formed from the l-dopa side chain. Thus through-space and through-bond 13C-13C connectivities were evaluated for C. neoformans melanins derived from the following isotopically enriched feedstocks: (a) d-[U-13C6]glucose and l-[12C]dopa (described under “Aliphatic Molecular Scaffolds for C. neoformans Melanin Pigment Deposition”); (b) d-[12C]glucose and l-[2,3-13C2]dopa reported previously (8) and re-examined herein using current experimental methodologies; and (c) dual-labeled d-[U-13C6]glucose and l-[2,3-13C2]dopa. As noted in conjunction with the experiments using ring-labeled l-dopa (see under “Spatial Proximity and Covalent Bonding of C. neoformans Melanin Molecular Structures Derived from Glucose and l-Dopa Benzenoid Moieties”), Fig. 3 shows that the purple 2D contours of sample a are dominated by 13C-enriched (oxy)alkane, alkene, carboxylate, and amide moieties. By comparison, Figs. 5 and and66 show that our 2D 13C-13C DARR for l-[2,3-13C2]dopa C. neoformans melanin with either d-[12C]glucose (sample b, blue contours) or d-[U-13C6]glucose (sample c, red contours) display a variety of cross-peaks from isotopically enriched aromatic and aliphatic groups. Table 1 again provides a guide to the chemical shift assignments that underlie our structural interpretations.

FIGURE 5.
Solid-state 2D through-space 13C-13C correlation spectra acquired at a 1H frequency of 750 MHz with 15 kHz MAS for C. neoformans melanins produced in the 24067 fungal cell line. A, 1D CPMAS 13C NMR spectra for C. neoformans melanin produced with l-[2,3- ...
FIGURE 6.
Solid-state NMR at 15 kHz MAS for C. neoformans melanin produced in the 24067 fungal cell line with l-[2,3-13C2]dopa and [U-13C6]glucose. Left, 2D contour plot from a 500-ms 13C-13C DARR experiment (35, 36) conducted at a 1H frequency of 600 MHz, with ...

The high field DARR experiments on C. neoformans melanins from d-[12C]glucose and l-[2,3-13C2]dopa showed many through-space proximities within the aromatic pigment core as well as cross-peaks between resonances assigned to ester, pyrrole-in-indole, oxymethylene, and alkyl chain pairs of moieties (170 × 125 and 55 × 30 ppm, Fig. 5) (9). In addition to supporting prior low-field proton-driven 13C-13C spin diffusion experiments showing connections within the aromatic core (8), the 55 × 30 ppm pairwise spatial proximity revealed in these high sensitivity 750 MHz 2D spectra offered particular support for proposed uncyclized or cleaved ring aliphatic molecular fragments associated with catecholamine-derived melanin pigments (3, 8, 20, 24).

As noted above, the d-[U-13C6]glucose-enriched sample a displayed similarly located DARR cross-peaks (171 × 126 ppm, Fig. 3), indicating ~6 Å spatial proximity between chitin amide and alkene groups. By comparison, DARR results for the dual-labeled sample c included spatial interactions between two pairs of carbons in these respective spectral regions, 171 × 127 and 169 × 117 ppm (Fig. 6). Whereas the first pair of cross-peaks coincided with features observed for the singly labeled C. neoformans melanins, the second pair was observed only for the sample with 13C-enriched glucose and l-dopa feedstocks. A provisional structural identification could be made using prior knowledge of the pigment and fungal cell-wall constituents (45), NMR of model compounds, and empirically based spectral predictions of various aromatic fragments (3, 9, 53). Thus, we attributed the 169 × 117 ppm feature to a through-space proximity between a glyceride carboxylate and/or a chitin amide and a free pyrrole, i.e. to distinct molecular constituents of the C. neoformans melanin assembly.

Our 2D through-bond SAR-COSY results (Figs. 5 and and6)6) provided a means to confirm the two-component assignment of the 169 × 117 ppm DARR cross-peak and to test whether the connections between chemical constituents of the dual-labeled C. neoformans melanin sample correspond to covalently bonded pairs. Significant through-bond SAR-COSY connections in l-[2,3-13C2]dopa C. neoformans melanin were observed only between rigid aromatic carbons (128 × 111 ppm, Fig. 5), whereas d-[U-13C6]glucose-enriched C. neoformans melanin produced no SAR-COSY signals at all in this spectral region (Fig. 3). Therefore, the bonded interactions involving amide/carboxylate carbons at ~168 ppm with aromatic and alkene groups resonating between 110 and 130 ppm (Fig. 6, green ovals) in the dual-labeled sample were taken to originate from different macromolecular components.

In terms of molecular architecture, the SAR-COSY results revealed covalent bonds between different pyrrole ring carbons (130 × 115 ppm) and between amide/carboxylate groups and free pyrroles ~(168 × 120 ppm). Thus, whereas the pyrrole SAR-COSY and DARR data of Fig. 6 bolstered the aromatic ring stacking proposal introduced under “Spatial Proximity and Covalent Bonding of C. neoformans Melanin Molecular Structures Derived from Glucose and l-Dopa Benzenoid Moieties,” the 2D NMR results also suggested carbon-containing covalent linkages between N-acetylglucosamines of chitin or membrane glycerides and the pigment pyrroles (Fig. 7). Among the possible interacting fungal cell-wall constituents that are reported to impact melanization are chitin and chitosan (44), which display amide resonances with the requisite 13C chemical shift values (39) and are thus strong candidates for bonded partners with the pigment pyrroles.

FIGURE 7.
Working model and architectural elements of C. neoformans melanins, incorporating established information on the cell wall (45, 60) and pairwise spatial and covalent connections derived from 2D solid-state NMR of isotopically enriched precursors. Schematics ...

Discussion

This study reveals several key design attributes of the C. neoformans melanin assembly and allows us to test previously proposed structural models (1, 3, 48,52) in related macromolecular systems. First, our 13C NMR spectra establish a sequence involving early (4 days) deposition of an aliphatic framework followed by later (14 days) appearance of an aromatic component (Fig. 1), suggesting that the cellular materials serve as a scaffolding for the developing pigment. As the pigment granules are deposited on this structural support, the aliphatic framework becomes chemically functionalized, permitting it to survive degradative procedures used to isolate the melanin ghosts and remain available for physical scrutiny. The increasing aromatic pigment buildup that is apparent in the spectroscopic data provides a molecular rationale for the augmented negative charge and cell hydrophobicity associated with C. neoformans and Aspergillus fumigatus melanization (28, 54). Moreover, the NMR-monitored growth in the proportion of aromatic pigment constituents with respect to the aliphatic scaffold in l-dopa and dopamine C. neoformans melanin preparations offers a structural explanation for previously reported increases in wall thickness and decreases in porosity in C. neoformans ghosts after 4, 7, and 10 days of growth, respectively (55).

Importantly, this study sheds new light on both the fungal scaffolding architecture and the pigment organization. Our evidence for proximal and bonded 13C-13C pairs supports an acid-resistant aliphatic scaffold involving interacting glucan, chitin, and acylglyceride architectural elements (Fig. 7). Previous scanning, transmission, and atomic force microscopy studies of melanized C. neoformans ghosts have been accommodated by a model in which pigment granules 50–80 nm in diameter are assembled into ~200-nm thick concentric layers that form a barrier against animal hosts and environmental challenges (28, 55). Whereas the formation of small granules rather than a contiguous melanin layer would demand a scaffold to anchor the particles, the layered pigment arrangement also underscores the prospect of both radial and axial particle growth during C. neoformans melanization. Here, we addressed the question of whether the granules are held together by e.g. cross-links or nonpigment scaffolding using 2D solid-state NMR experiments on isotopically enriched fungal eumelanins.

The isotopically labeled benzenoid aromatic rings of the pigment produced from l-[ring-13C6]dopa and [U-13C6]glucose exhibit a network of short range spatial proximities (up to ~6 Å) (46), including several spin connectivities to the aliphatic framework. This C. neoformans melanin exhibits several overlapping aromatic carbon covalent connections in 2D NMR but no direct one-bond aromatic-aliphatic connections. The observed proximity and bonding constraints between pairs of intact benzenoid aromatic sites reinforce prior proposals of planar porphyrin-like oligomeric clusters deduced from x-ray diffraction, scanning tunneling microscopy, optical spectroscopy, and density functional theory (48,50). Moreover, a porphyrin-like arrangement should favor the predominantly aromatic-aromatic interactions observed among the longer distance cross-peaks in the 500-ms DARR spectrum.

The benzenoid-mediated interactions revealed by 2D NMR also support the energetically favored π-stacking of aromatic structures predicted computationally (51, 52) and account for magnetically distinct but overlapping aromatic resonances in the solid-state 13C (8, 9, 19, 20) and 2D 13C-15N (24) NMR spectra. Such aromatic interactions can produce the layered architecture observed by transmission electron microscopy (28, 55), also providing a rationale for the observation of negative particle charge and hydrophobic character associated with melanization (28, 54). The increasing dispersion of indole-based NMR spectral features observed at day 14 also suggests a range of similar aromatic environments that could reflect imperfect alignment of successive layers of stacked aromatic ring structures (Fig. 7). Such heterogeneous organization could result in a material that is locally ordered but globally amorphous in structure and thus unsuitable for crystallographic analysis. Furthermore, progressive melanization could account in turn for the observed constriction of pores in melanin ghosts, which has been proposed to prevent infusion of echinocandin antifungal drugs while still permitting nutrient passage into the fungal cells (4). Our solid-state NMR data thus show that the indole-based aromatic rings are interconnected predominantly as postulated previously (48), and additionally that their benzenoid aromatic structures can be proposed as associating “hydrophobically” rather than being linked covalently to the long-chain acylglycerides of the underlying aliphatic scaffolding.

By contrast, the labeled pyrrole aromatic structures (often within indole units in C. neoformans melanins) that are derived from l-[2,3-13C2]dopa exhibit both spatial and bonded connections to aliphatics originating from [U-13C6]glucose. Different pyrrole ring sites (130 × 115 ppm) are linked to each other and also to carboxylate- or amide-containing structures from other cellular constituents. The former linkage is in accord with a close 13C-15N spatial relationship reported recently in cell-free l-dopa melanin (24). The latter molecular pair ~(169 × 117 ppm) is attributable to cellular constituents such as chitin or acylglycerides and to free pyrrole pigment units, respectively (Fig. 7). As described for the l-[ring-13C6]dopa melanins, the pyrrole-in-indole aromatic ring connections in solid-state NMR of l-[2,3-13C2]dopa C. neoformans melanin support the postulated stacking of indole-based aromatic units and covalent cross-linking between such aromatic moieties (48). Moreover, the intercomponent pyrrole attachment to amides in N-acetylglucosamines of chitin or carboxylates in membrane glycerides suggests covalent modification of the cell membrane, chitin, or chitosan components (44) to form a progressively more complex macromolecular assembly during C. neoformans melanization (Figs. 1 and and5).5). Thus, whereas the pyrrole SAR-COSY and DARR data of Figs. 5 and and66 bolstered the aromatic ring stacking proposal introduced under “Spatial Proximity and Covalent Bonding of C. neoformans Melanin Molecular Structures Derived from Glucose and l-Dopa Benzenoid Moieties,” the 2D NMR data also suggested carbon-containing covalent linkages between the pigment pyrroles and either chitin-derived N-acetylglucosamines or membrane glycerides.

Chitin, for instance, is a major hydrophilic cell-wall constituent that forms hydrogen-bonded microfibrils to confer mechanical strength and is reported to interact strongly with melanin in A. nidulans (45). In Candida albicans, chitin is essential for externalization of melanin (56); in C. neoformans, both chitin synthase (57) and chitosan in the cell wall (44) are reported to modulate melanization. Chitin and melanin have also been reported to interact closely in marine invertebrates (58) and insect wings (59). Hence, the covalent connection between chitin and melanin suggested by this spectroscopic study is supported by, and consistent with, independent observations in animals and fungi showing these components to be closely linked. The versatility of the C. neoformans melanin pigment assembly that allows for both stacking and scaffold binding reflects its dual hydrophobic-hydrophilic character; our conceptual model posits that the hydrophobic aromatics assemble in multiple layers, and the hydrophilic moieties “reach out” to bind covalently with cellular glycerides or polysaccharides such as chitin or chitosan. Given the commonality of aromatic core structures demonstrated recently for synthetic and C. neoformans melanins (24), these defining arrangements also help to establish the architectural capabilities of designed melanin-based soft materials.

In conclusion, the current solid-state NMR findings offer the first biophysical view of the mechanism by which melanin is assembled in the fungal cell wall, providing a structural rationale for the major pigment-associated changes in melanized C. neoformans cell-wall architecture probed previously using diverse physical characterization methods (4). The 1D 13C and 2D 13C-13C solid-state NMR results support the following picture: an early developing aliphatic scaffold consisting of several proximal polysaccharides and acylglycerides; a late-developing aromatic pigment, including indole and pyrrole structural moieties; and architectural networks that include close indole-indole associations and pyrrole-chitin covalent bonds. The progressive development of multicomponent melanized assemblies, wherein aromatic rings are nevertheless predominantly proximal to each other, supports proposed interlayer stacking (48,52) or lateral interactions and argues for a striking degree of regulation accompanying this particular free radical polymerization process. Although this work focuses on the formation and assembly of intact melanin pigments in fungal cells, our insights into the structural requisites of melanin-cell wall assemblies should have broader practical potential as follows: for enhancing the efficacy of melanoma treatments; bioremediation of radioactively contaminated soils or reactors, and design of bioinspired materials for protective coatings and therapeutic drug delivery (2,4).

Acknowledgments

The 600 MHz NMR facilities used in this work are operated by City College and the City University of New York Institute for Macromolecular Assemblies, with additional infrastructural support provided by National Institutes of Health Grant 8G12 MD007603-29 from the National Institute on Minority Health and Health Disparities. We thank Dr. Hsin Wang for technical support and the reviewers for helpful suggestions related to 2D NMR interpretation and literature citations.

*This work was supported, in whole or in part, by National Institutes of Health Grant R01-AI052733. This work was also supported by a STAR Center Grant from the New York State Office of Science, Technology, and Academic Research for the 750 MHz NMR experiments (to New York Structural Biology Center).

3The abbreviations used are:

CPMAS
cross-polarization magic-angle spinning NMR
DARR
dipolar assisted rotational resonance
SAR-COSY
sensitive absorptive refocused scalar correlation spectroscopy
CP
cross-polarization
MAS
magic-angle spinning.

References

1. d'Ischia M., Napolitano A., Pezzella A., Meredith P., Sarna T. (2009) Chemical and structural diversity in eumelanins: unexplored bio-optoelectronic materials. Angew. Chem. Int. Ed. Engl. 48, 3914–3921 [PMC free article] [PubMed]
2. Simon J. D., Peles D. N. (2010) The red and the black. Acc. Chem. Res. 43, 1452–1460 [PubMed]
3. Della Vecchia N. F., Avolio R., Alfè M., Errico M. E., Napolitano A., d'Ischia M. (2013) Building-block diversity in polydopamine underpins a multifunctional eumelanin-type platform tunable through a quinone control point. Adv. Funct. Mater. 23, 1331–1340
4. Eisenman H. C., Casadevall A. (2012) Synthesis and assembly of fungal melanin. Appl. Microbiol. Biotechnol. 93, 931–940 [PMC free article] [PubMed]
5. Coelho C., Bocca A. L., Casadevall A. (2014) The tools for virulence of cryptococcus neoformans. Adv. Appl. Microbiol. 87, 1–41 [PubMed]
6. Schnitzer M., Chan Y. K. (1986) Structural characteristics of a fungal melanin and a soil humic acid. Soil Sci. Soc. Am. J. 50, 67–71
7. Duff G. A., Roberts J. E., Foster N. (1988) Analysis of the structure of synthetic and natural melanins by solid-phase NMR. Biochemistry 27, 7112–7116 [PubMed]
8. Tian S., Garcia-Rivera J., Yan B., Casadevall A., Stark R. E. (2003) Unlocking the molecular structure of fungal melanin using 13C biosynthetic labeling and solid-state NMR. Biochemistry 42, 8105–8109 [PubMed]
9. Adhyaru B. B., Akhmedov N. G., Katritzky A. R., Bowers C. R. (2003) Solid-state cross-polarization magic-angle spinning 13C and 15N NMR characterization of sepia melanin, sepia melanin free acid, and human hair melanin in comparison with several model compounds. Magn. Reson. Chem. 41, 466–474
10. Thureau P., Ziarelli F., Thévand A., Martin R. W., Farmer P. J., Viel S., Mollica G. (2012) Probing the motional behavior of eumelanin and pheomelanin with solid-state NMR spectroscopy: new insights into the pigment properties. Chemistry 18, 10689–10700 [PubMed]
11. Kim S. J., Schaefer J. (2008) Hydrophobic side-chain length determines activity and conformational heterogeneity of a vancomycin derivative bound to the cell wall of Staphylococcus aureus. Biochemistry 47, 10155–10161 [PMC free article] [PubMed]
12. Renault M., Cukkemane A., Baldus M. (2010) Solid-state NMR spectroscopy on complex biomolecules. Angew. Chem. Int. Ed. Engl. 49, 8346–8357 [PubMed]
13. Dick-Pérez M., Zhang Y., Hayes J., Salazar A., Zabotina O. A., Hong M. (2011) Structure and interactions of plant cell-wall polysaccharides by two-and three-dimensional magic angle-spinning solid-state NMR. Biochemistry 50, 989–1000 [PubMed]
14. Kim S. J., Singh M., Preobrazhenskaya M., Schaefer J. (2013) Staphylococcus aureus peptidoglycan stem packing by rotational-echo double resonance NMR spectroscopy. Biochemistry 52, 3651–3659 [PMC free article] [PubMed]
15. Takahashi H., Ayala I., Bardet M., De Paëpe G., Simorre J.-P., Hediger S. (2013) Solid-state NMR on bacterial cells: selective cell-wall signal enhancement and resolution improvement using dynamic nuclear polarization. J. Am. Chem. Soc. 135, 5105–5110 [PubMed]
16. Serra O., Chatterjee S., Figueras M., Molinas M., Stark R. E. (2014) Deconstructing a plant macromolecular assembly: chemical architecture, molecular flexibility, and mechanical performance of natural and engineered potato suberins. Biomacromolecules 15, 799–811 [PMC free article] [PubMed]
17. Wang Y., Aisen P., Casadevall A. (1996) Melanin, melanin “ghosts,” and melanin composition in Cryptococcus neoformans. Infect. Immun. 64, 2420–2424 [PMC free article] [PubMed]
18. Rosas A. L., Nosanchuk J. D., Feldmesser M., Cox G. M., McDade H. C., Casadevall A. (2000) Synthesis of polymerized melanin by Cryptococcus neoformans in infected rodents. Infect. Immun. 68, 2845–2853 [PMC free article] [PubMed]
19. Zhong J., Frases S., Wang H., Casadevall A., Stark R. E. (2008) Following fungal melanin biosynthesis with solid-state NMR: biopolymer molecular structures and possible connections to cell-wall polysaccharides. Biochemistry 47, 4701–4710 [PubMed]
20. Chatterjee S., Prados-Rosales R., Frases S., Itin B., Casadevall A., Stark R. E. (2012) Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors. Biochemistry 51, 6080–6088 [PMC free article] [PubMed]
21. Stark R. E., Yu B., Zhong J., Yan B., Wu G., Tian S. (2013) Environmental NMR: high resolution magic angle spinning. eMagRes. 2, 377–388
22. Crescenzi O., Kroesche C., Hoffbauer W., Jansen M., Napolitano A., Prota G., Peter M. G. (1994) Synthesis of dopamines labelled with 13C in the α or β-side chain position and their application to structural studies on melanins by solid-state NMR spectroscopy. Liebigs Ann. Chem. 1994, 563–567
23. d'Ischia M., Wakamatsu K., Napolitano A., Briganti S., Garcia-Borron J.-C., Kovacs D., Meredith P., Pezzella A., Picardo M., Sarna T., Simon J. D., Ito S. (2013) Melanins and melanogenesis: methods, standards, protocols. Pigment Cell Melanoma Res. 26, 616–633 [PubMed]
24. Chatterjee S., Prados-Rosales R., Tan S., Itin B., Casadevall A., Stark R. E. (2014) Demonstration of a common indole-based aromatic core in natural and synthetic eumelanins by solid-state NMR. Org. Biomol. Chem. 12, 6730–6736 [PMC free article] [PubMed]
25. Ito S. (2003) A chemist's view of melanogenesis. Pigment Cell Res. 16, 230–236 [PubMed]
26. Nosanchuk J. D., Casadevall A. (2003) Budding of melanized Cryptococcus neoformans in the presence or absence of l-dopa. Microbiology 149, 1945–1951 [PubMed]
27. Eisenman H. C., Frases S., Nicola A. M., Rodrigues M. L., Casadevall A. (2009) Vesicle-associated melanization in Cryptococcus neoformans. Microbiology 155, 3860–3867 [PMC free article] [PubMed]
28. Garcia-Rivera J., Eisenman H. C., Nosanchuk J. D., Aisen P., Zaragoza O., Moadel T., Dadachova E., Casadevall A. (2005) Comparative analysis of Cryptococcus neoformans acid-resistant particles generated from pigmented cells grown in different laccase substrates. Fungal Genet. Biol. 42, 989–998 [PubMed]
29. Folch J., Lees M., Sloane Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509 [PubMed]
30. Morcombe C. R., Zilm K. W. (2003) Chemical shift referencing in MAS solid state NMR. J. Magn. Reson. 162, 479–486 [PubMed]
31. Metz G., Wu X. L., Smith S. O. (1994) Ramped-amplitude cross-polarization in magic angle-spinning NMR. J. Magn. Reson. Ser. A 110, 219–227
32. Bennett A. E., Rienstra C. M., Auger M., Lakshmi K. V., Griffin R. G. (1995) Heteronuclear decoupling in rotating solids. J. Chem. Phys. 103, 6951–6958
33. Fung B. M., Khitrin A. K., Ermolaev K. (2000) An improved broadband decoupling sequence for liquid crystals and solids. J. Magn. Res. 142, 97–101 [PubMed]
34. Johnson R. L., Schmidt-Rohr K. (2014) Quantitative solid-state 13C NMR with signal enhancement by multiple cross-polarization. J. Magn. Reson. 239, 44–49 [PubMed]
35. Takegoshi K., Nakamura S., Terao T. (2001) 13C-1H dipolar assisted rotational resonance in magic angle spinning NMR. Chem. Phys. Lett. 344, 631–637
36. Morcombe C. R., Gaponenko V., Byrd R. A., Zilm K. W. (2004) Diluting abundant spins by isotope edited radio frequency field assisted diffusion. J. Am. Chem. Soc. 126, 7196–7197 [PubMed]
37. States D. J., Haberkorn R. A., Ruben D. J. (1982) A two-dimensional nuclear Overhauser experiment with pure absorption phase in four quadrants. J. Magn. Reson. 48, 286–292
38. Lee D., Struppe J., Elliott D. W., Mueller L. J., Titman J. J. (2009) Sensitive absorptive refocused scalar correlation NMR spectroscopy in solids. Phys. Chem. Chem. Phys. 11, 3547–3553 [PubMed]
39. Fukamizo T., Kramer K. J., Mueller D. D., Schaefer J., Garbow J., Jacob G. S. (1986) Analysis of chitin structure by nuclear magnetic resonance spectroscopy and chitinolytic enzyme digestion. Arch. Biochem. Biophys. 249, 15–26 [PubMed]
40. Li K. L., Tihal C. A., Guo M., Stark R. E. (1993) Multinuclear and magic angle spinning NMR investigations of molecular organization in phospholipid-triglyceride aqueous dispersions. Biochemistry 32, 9926–9935 [PubMed]
41. Kao P. F., Wang S. H., Hung W. T., Liao Y. H., Lin C. M., Yang W. B. (2012) Structural characterization and antioxidative activity of low-molecular-weights β-1,3-glucan from the residue of extracted Ganoderma lucidum fruiting bodies. J. Biomed. Biotechnol. 2012, 673764. [PMC free article] [PubMed]
42. Cabib E., Roh D. H., Schmidt M., Crotti L. B., Varma A. (2001) The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J. Biol. Chem. 276, 19679–19682 [PubMed]
43. Perkins S. J., Johnson L. N., Phillips D. C., Dwek R. A. (1977) High resolution 1H- and 13C-NMR spectra of d-glucopyranose, 2-acetamido-2-deoxy-d-glucopyranose, and related compounds in aqueous media. Carbohydr. Res. 59, 19–34
44. Baker L. G., Specht C. A., Donlin M. J., Lodge J. K. (2007) Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans. Eukaryot. Cell 6, 855–867 [PMC free article] [PubMed]
45. Free S. J. (2013) Fungal cell wall organization and biosynthesis Adv. Genet. 81, 33–82 [PubMed]
46. Crocker E., Patel A. B., Eilers M., Jayaraman S., Getmanova E., Reeves P. J., Ziliox M., Khorana H. G., Sheves M., Smith S. O. (2004) Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin. J. Biomol. NMR 29, 11–20 [PubMed]
47. Chen L., Olsen R. A., Elliott D. W., Boettcher J. M., Zhou D. H., Rienstra C. M., Mueller L. J. (2006) Constant-time through-bond 13C correlation spectroscopy for assigning protein resonances with solid-state NMR spectroscopy. J. Am. Chem. Soc. 128, 9992–9993 [PubMed]
48. Zajac G. W., Gallas J. M., Cheng J., Eisner M., Moss S. C., Alvarado-Swaisgood A. E. (1994) The fundamental unit of synthetic melanin: a verification by tunneling microscopy of x-ray scattering results. Biochim. Biophys. Acta 1199, 271–278 [PubMed]
49. Cheng J., Moss S. C., Eisner M. (1994) X-ray characterization of melanins. II. Pigment Cell Res. 7, 263–273 [PubMed]
50. Tran M. L., Powell B. J., Meredith P. (2006) Chemical and structural disorder in eumelanins: a possible explanation for broadband absorbance. Biophys. J. 90, 743–752 [PubMed]
51. Meredith P., Sarna T. (2006) The physical and chemical properties of eumelanin. Pigment Cell Res. 19, 572–594 [PubMed]
52. Meng S., Kaxiras E. (2008) Theoretical models of eumelanin protomolecules and their optical properties. Biophys. J. 94, 2095–2105 [PubMed]
53. Johnson R. L., Anderson J. M., Shanks B. H., Fang X., Hong M., Schmidt-Rohr K. (2013) Spectrally edited two-dimensional 13C-13C NMR spectra without diagonal ridge for characterizing 13C-enriched low-temperature carbon materials. J. Magn. Reson. 234, 112–124 [PubMed]
54. Pihet M., Vandeputte P., Tronchin G., Renier G., Saulnier P., Georgeault S., Mallet R., Chabasse D., Symoens F., Bouchara J.-P. (2009) Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia. BMC Microbiol. 9, 177. [PMC free article] [PubMed]
55. Eisenman H. C., Nosanchuk J. D., Webber J. B., Emerson R. J., Camesano T. A., Casadevall A. (2005) Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans. Biochemistry 44, 3683–3693 [PubMed]
56. Walker C. A., Gómez B. L., Mora-Montes H. M., Mackenzie K. S., Munro C. A., Brown A. J., Gow N. A., Kibbler C. C., Odds F. C. (2010) Melanin externalization in Candida albicans depends on cell-wall chitin structures. Eukaryot. Cell 9, 1329–1342 [PMC free article] [PubMed]
57. Walton F. J., Idnurm A., Heitman J. (2005) Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol. Microbiol. 57, 1381–1396 [PubMed]
58. Hwang D. S., Masic A., Prajatelistia E., Iordachescu M., Waite J. H. (2013) Marine hydroid perisarc: a chitin- and melanin-reinforced composite with DOPA-iron(III) complexes. Acta Biomater. 9, 8110–8117 [PubMed]
59. Stavenga D. G., Leertouwer H. L., Hariyama T., De Raedt H. A., Wilts B. D. (2012) Sexual dichromatism of the damselfly Calopteryx japonica caused by a melanin-chitin multilayer in the male wing veins. PLoS One 7, e49743. [PMC free article] [PubMed]
60. Hardison S. E., Brown G. D. (2012) C-type lectin receptors orchestrate antifungal immunity. Nat. Immunol. 13, 817–822 [PMC free article] [PubMed]
61. Garbow J. R., Ferrantello L. M., Stark R. E. (1989) 13C nuclear magnetic resonance study of suberized potato cell wall. Plant Physiol. 90, 783–787 [PubMed]
62. Pascoal Neto C., Rocha J., Gil A., Cordeiro N., Esculcas A. P., Rocha S., Delgadillo I., de Jesus J. D., Correia A. J. (1995) 13C solid-state nuclear magnetic resonance and Fourier transform infrared studies of the thermal decomposition of cork. Solid State Nucl. Magn. Reson. 4, 143–51 [PubMed]
63. Atalla R. H., Gast J. C., Sindorf D. W., Bartuska V. J., Maciel G. E. (1980) 13C NMR spectra of cellulose polymorphs. J. Am. Chem. Soc. 102, 3249–3251
64. Earl W. L., VanderHart D. L. (1980) High resolution, magic angle sampling spinning carbon-13 NMR of solid cellulose I. J. Am. Chem. Soc. 102, 3251–3252

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology