The complete assignment of the proton chemical shifts obtained by nuclear magnetic resonance (NMR) spectroscopy of de-O-acetylated glucuronoxylomannans (GXMs) from Cryptococcus neoformanspermitted the high-resolution determination of the total structure of any GXM. Six structural motifs based on an α-(1→3)-mannotriose substituted with variable quantities of 2-O-β- and 4-O-β-xylopyranosyl and 2-O-β-glucopyranosyluronic acid were identified. The chemical shifts of only the anomeric protons of the mannosyl residues served as structure reporter groups (SRG) for the identification and quantitation of the six triads present in any GXM. The assigned protons for the mannosyl residues resonated at clearly distinguishable positions in the spectrum and supplied all the information essential for the assignment of the complete GXM structure. This technique for assigning structure is referred to as the SRG concept. The SRG concept was used to analyze the distribution of the six mannosyl triads of GXMs obtained from 106 isolates of C. neoformans. The six mannosyl triads occurred singularly or in combination with one or more of the other triads. The identification and quantitation of the SRG were simplified by using a computer-simulated artificial neural network (ANN) to automatically analyze the SRG region of the one-dimensional proton NMR spectra. The occurrence and relative distribution of the six mannosyl triads were used to chemotype C. neoformans on the basis of subtle variations in GXM structure determined by analysis of the SRG region of the proton NMR spectrum by the ANN. The data for the distribution of the six SRGs from GXMs of 106 isolates of C. neoformansyielded eight chemotypes, Chem1 through Chem8.

Cryptococcus neoformans was cultured in a chemically defined medium. The culture was adjusted to 0.25% formaldehyde or autoclaved after 5 days of growth at 35C, and a cell-free supernatant was obtained by centrifugation. Solid calcium acetate was added to the supernatant to give a 5% solution, and the pH was adjusted to-5 with glacial acetic acid. The polysaccharide (PS) was precipitated by the addition of 3 volumes of 95% ethanol. The PS was dissolved in 0.2 M NaCI, and insoluble calcium salts were solubilized by the addition of several drops of glacial acetic acid. The PS solution was treated by ultrasonic irradiation for 15 min. This concurrently decreased the molecular weight of the PS and reduced the viscosity of the solution. The ultrasonically irradiated PS was precipitated by differential complexation with hexadecyltrimethylammonium bromide at 23°C, the complex was dissolved in 1 M NaCl, and the glucuronoxylomannan was precipitated by adding 3 volumes of ethanol. The glucuronoxylomannan was dissolved in 1 M NaCl and then ultrasonically irradiated for 2 h to reduce the molecular mass to a limiting value of-100 kDa (GXMS). The purified GXMS was centrifuged, dialyzed, and finally recovered by lyophilization. GXMS was chromatographed on DEAEcellulose at reasonable concentrations without the complication of high solution viscosity. The sugar composition and structure of GXMS were determined by gas-liquid chromatography, permethylation gas-liquid chromatography-mass spectrometry, and 13C nuclear magnetic resonance spectroscopy. The improved solution characteristics of GXMS were ideal for the determination of its chemical and serological properties.

Roundabout 1, or Robo1, is a cell surface signaling molecule important in axon guidance. Its interaction with heparan sulfate (HS) and members of the Slit protein family is essential to its activity, making characterization of these interactions by structural methods, such as NMR, highly desirable. However, the fact that Robo1 is a glycosylated protein prevents employment of commonly used bacterial hosts for expression of properly glycosylated forms with the uniform 15N, 13C, and 2H labeling needed for NMR studies. Here, we apply an alternative methodology, based on labeling with a single amino acid type and high structural content NMR data, to characterize a two-domain construct of glycosylated Robo1 (Robo1–Ig1–2) interacting with a synthetic HS tetramer (IdoA-GlcNS6S-IdoA2S-GlcNS6S-(CH2)5NH2). Significant chemical shift perturbations of the crosspeak from K81 on titration with the tetramer provide initial evidence for the location of a binding site and allow determination of a 255 μM disassociation constant. The binding epitopes, bound conformation, and binding site placement of the HS tetramer have been further characterized by saturation transfer difference (STD), transferred nuclear Overhauser effect (trNOE), and paramagnetic perturbation experiments. A model of the complex has been generated using constraints derived from the various NMR experiments. Postprocessing energetic analysis of this model provides a rationale for the role each glycan residue plays in the binding event, and examination of the binding site in the context of a previous Robo-Slit structure provides a rationale for modulation of Robo-Slit interactions by HS.

Capsular glucuronoxylomannans (GXM) of Cryptococcus neoformans var. neoformans isolates from patients with recurrent cryptococcal meningitis were analyzed by 1 H nuclear magnetic resonance spectroscopy and for reactivity with factor sera (Iatron, Tokyo, Japan). For each patient the initial and relapse isolates had previously been shown to be indistinguishable by DNA restriction fragment length polymorphism analysis. For patients J11 and J22 the GXM of the initial and relapse isolates were identical. For patients SB4 and SB6 the GXM of the initial and relapse isolates differed in structure and reactivity with factor sera. In patient SB4 the initial isolate had a serotype A/D structure, and the first relapse isolate had a serotype A structure. The second relapse isolate was a mixture of structures composed of serotype D components, glucuronomannan (GM), and a minor serotype A component. Analysis of the initial isolate from patient SB6 showed a structure composed mainly of serotype D, GM, and minor serotype A components and components not assigned to a particular serotype (N). The relapse isolate had the same composition as the initial isolate except for an increase in the serotype A component. This increase in the serotype A component of the relapse isolate resulted in a change in the serological specificity from serotype D to serotype A/D. The initial isolate from patient J9 had serotype D and GM structures. The first two relapse isolates had serotype D, N, and GM structures and a minor serotype A component. The third relapse isolate had mainly a serotype D structure. All the J9 isolates reacted only with serotype D-specific factor serum. These results indicate that some isolates obtained from patients with recurrent C. neoformans infections have undergone a change in GXM structure during the course of infection. The modification of GXM structure observed in some relapse isolates is reflected in changed serological properties. The results may have important implications for the design of vaccines and antibodybased therapeutic strategies against C. neoformans.

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Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In these cases, sparse isotopic labeling (single or small subsets of amino acids) combined with long range paramagnetic constraints and improved computational modeling offer an alternative. This perspective provides a brief overview of this approach and two discussions of potential applications; one involving a very large system (an Hsp90 homolog) in which perdeuteration is possible and methyl-TROSY sequences can potentially be used to improve resolution, and one involving ligand placement in a glycosylated protein where resolution is achieved by single amino acid labeling (the sialyltransferase, ST6Gal1). This is not intended as a comprehensive review, but as a discussion of future prospects that promise impact on important questions in the structural biology area.

The repetitive DNA sequence poly[d(GT).d(CA)], (polyGT) can be used to generate DNA fingerprints that distinguish different yeast genera. In this study we demonstrate that the probe can also be used to distinguish individual strains of clinical isolates of Candida albicans. Isolates were fingerprinted by probing Southern blots of restriction enzyme-cleaved DNA samples with radioactively labelled polyGT. The discrimination between strains was clearer than can be achieved by direct visualization of ethidium bromide stained gels and was comparable to that achieved with previous DNA probes. However, the advantage of this probe is that it is not limited to Candida species since polyGT sequences appear to be ubiquitous in eukaryotes. Fingerprints were also generated from Southern blots using a commercial (AMBIS) radioanalytical imaging computer system. A proprietary software package (MICRO PM) was effective in discriminating between the C. albicans strains. These preliminary results indicate the potential value of this probe for discrimination between Candida isolates in epidemiological studies of candidosis.The increased incidence of systemic forms of candidosis in immunocompromised patients, and of mucosal and oesophageal candidosis in AIDS patients, has emphasized the need for a rapid typing system for epidemiological studies. DNA fingerprinting techniques are particularly attractive as the assessment of genotype generally involves less subjective judgement than the assessment of phenotype.Conserved repetitive sequences present in the genome of Candida species, such as the ribosomal RNA genes [5] and mitochondrial DNA [8] have already been shown to give rise to recognizably different patterns in ethidium bromide stained gels of restriction enzyme-cleaved DNA. For example, ethidium bromide stained gels of EcoRi digested DNA have been successfully used to follow outbreaks of systemic candidosis [6]; other studies have suggested that Hinfl can be an even more effective restriction enzyme for identifying Candida albicans strains by this type of restriction fragment length polymorphism (RFLP) analysis [12]. There are, however drawbacks to this approach; first, the patterns can be difficult to distinguish against the background smear of other genomic sequences and second, RFLPs detectable within all copies of a repeated sequence will reflect relatively ancient strain divergences where mutations have been fixed. When strains have diverged more recently, for example within hospi-

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