Results
Diffraction studies on whole cells Our initial studies were designed to ascertain whether we could
gain insight into melanin structure from pigment in the cell wall.
Hence, we compared the X-ray diffraction features observed with
melanized and non-melanized whole C. neoformans cells using 1.54-
Angstrom source. The scattering of X-rays by crystalline structures
produces sharp peaks in the diffraction spectrum that serve as a
signature for the crystal analyzed. In contrast, amorphous
compounds like melanin and polysaccharide produce broad
features in the diffraction spectrum known as non-Bragg features
resulting from the absence of coherent scattering from regular and
repeating structures (e.g., crystals). When whole cells were
analyzed the essential feature of these measurements was the
presence of a broad non- Bragg feature, whose location was
identified in units of Q (reciprocal angstroms). The parameter Q
provides a measure of the spacing for the diffracting structure
(Figure 1). To explore the origin of this signal we compared the
diffraction spectrum of encapsulated and acapsular C. neoformans
cells in their melanized and non-melanized states (Figure 1). The
acapsular strain samples, whether melanized or non-melanized
produced a spectrum that lacked the amorphous peak but instead
showed several crystalline Bragg peaks (Figure 1). Since the
position of these peaks in the melanized and non-melanized strains
was the same we conclude that they do not come from melanin.
Next we compared the diffraction pattern of whole encapsulated
cells, polysaccharide (PS) naturally released from such cells (ExoPS)
and polysaccharide extracted from the capsules (Cap-PS) using
dimethethyl sulfoxide (DMSO) from four different cryptococcal
Fungal Melanins Differ in Stacking Distances
PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e30299
strains differing in serotype and polysaccharide structure [Table 1].
Whole cells produced spectra with two Q values, referred to as Q1
and Q2, in the range of 1.38–1.39 and 1.48–1.51, respectively (see
Figure 2 for representative spectra). In contrast, the diffraction
spectra of isolated exopolysaccharide and polysaccharides released
from the capsule was in the range 1.46–1.51, a value consistent
with the range for Q2. Numerous Bragg-type diffraction peaks
were apparent in all samples studied. Since these peaks occur in
the same position irrespective of the presence of capsule or
melanin we attribute their origin from microcrystalline impurities.
The presence of melanin in the polysaccharide preparation was
ruled out by the absence of any pigmentation or ESR signal
indicative of a free radical population (data not shown).