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RESEARCH PROFILES Hitting the magic angle for cell wall analysis
F1 (ppm)
F1 (ppm)
HR-MAS NMR spectra. The combined Many bacteria shield themselves from the protein solutions didn’t work well with use of 3-D hydrogen–carbon–carbon– magic angle spinning, so we had to eximmune system and therapeutics with an hydrogen total correlation spectroscopy plore what combination of methods elaborate cell wall. For example, mem(HCCH-TOCSY) and correlation specwould work best with suspended bactebers of the genus Mycobacterium, which troscopy (HCCH-COSY) produces rial samples,” he explains. includes the organisms responsible for good resolution of proton- and 13CFinding the optimal experimental causing tuberculosis and leprosy, possess conditions involved studying the effects labeled sugar units in the bacterial polya cell wall rich in polysaccharides and saccharides. “Since each sugar unit forms long-chain fatty acids. Understanding the of varying the NMR rotor speed, trying various pulse sequences, and altering a complete spin system separated from makeup of these walls is important for temperature and cell density. The investi- the next spin system by oxygen at the drug discovery efforts and is typically linkage, these individual done using time-honored spin systems are easily —and time-consuming— (f) (b) (e) (a) identified in the HCCHmethods of degradative TOCSY and HCCHanalysis. COSY spectra,” says Li. To hasten the analysis The researchers then and to provide a means of used the growing literature rapidly assessing the ef(h) (g) (d) (c) of published polysaccharide fects that drugs and ge65 100 NMR spectra, supplementnetic changes have on cell ed by spectra they obtained wall integrity, Wei Li and 75 104 on purified mycobacterial colleagues at the Universicell wall components, to ty of Tennessee (UT) 85 108 make the final assignments. Health Science Center F2 (ppm) F2 (ppm) 5.25 5.05 4.85 4.3 3.8 3.3 The complete analysis rehave developed a method HR-MAS NMR spectra of whole M. smegmatis cells at temperatures of 7, 17, 27, quires a mere 5–10 mg of of identifying key cell live cells grown in media wall components in intact and 37 °C. (a–d) Anomeric signals and (e–h) carbohydrate signals are shown. 13 with 0.2% [ C]-glycerol bacteria through the use 13 gators also examined the effects of labeland 0.2% [ C]-glucose as the main carof high-resolution magic angle spinning ing carbohydrate and lipid components bon sources, which Li says keeps the cost NMR (HR-MAS NMR). They demonby growing M. smegmatis with various of the method low and may allow testing strated this approach, which is described 13 C-enriched food sources. The goal of of single colonies from agar plates. Cell in the September 15 issue of Analytical Chemistry (pp 5785–5792), on the nonthis tinkering was to produce NMR spec- viability is >90% throughout the several pathogenic model organism Mycobactra with enough sensitivity and peak reso- hours of data acquisition needed to comlution to identify key carbohydrate and plete the NMR analysis. terium smegmatis. lipid components in the bacterial cell wall To demonstrate the technique’s utiliIn an earlier project, Li had used HRty, the researchers showed that they MAS NMR to search for reliable metabolic in the shortest amount of time. “Ultimately, we’d like to use NMR could use it to identify changes in the biomarkers in cancer cells. Then, Richard to screen for drug activity and study cell wall composition of two different Lee, a UT colleague who was in search of a nondestructive method for characterizing gene mutations, so we need the method mycobacteria dosed with the antibiotic to be not only fast, so that we can exethambutol (Glycobiology 2005, 15, mycobacteria, asked Li to run a sample. amine libraries of compounds, but sensi- 139–151). The next step is to add nuThe results from this initial collaboration tive enough so that we don’t need large were encouraging, and Li thought multiclear Overhauser effect NMR to the numbers of cells,” says Li. Sensitivity is dimensional whole-cell HR-MAS NMR protocol to determine where the various particularly important when working methods might provide the type of inforcell wall components reside in relation with mycobacteria, some of which are mation that Lee needed. to one another in live mycobacteria. slow growers and can be difficult to cul- “The interactions between cell wall Li’s first inclination was to combine ture in large amounts. the well-established pulse sequences decomponents [are] critical to cell wall The researchers eventually settled on veloped for analyzing protein structures function, and having a method to study with the HR-MAS techniques that work a four-pronged approach that enables these in vivo would be important for them to identify major and minor cell well with semisolid samples. “But many drug development efforts,” says Li. a wall components from the resulting pulse sequences intended for use with —Joe Alper O C T O B E R 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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