Analytical Currents: Identifying "generation gaps" among proteins

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ANALYTICAL CURRENTS Identifying “generation gaps” among proteins Many cellular structures are dynamic, with fresh proteins replacing older ones. It’s now possible to distinguish old and new proteins with a technique devised by Mark Ellisman, Roger Tsien, and colleagues at the University of California– San Diego and the University of Western Ontario (Canada). The method can be used with fluorescence microscopy and electron microscopy (EM), which has higher resolution. In the new technique, a tag containing four cysteines is genetically inserted (a)

(b)



(c)

30°

(d)

90°

3-D projections of gap junction plaques at various stages of refurbishing. (a–c) Younger proteins (red) gradually replace the older ones (green); in (d), the staining is reversed. (Adapted with permission. Copyright 2002 American Association for the Advancement of Science.)

nexin43. Able to distinguish younger from older proteins by color, the researchers saw that the younger proteins formed nearly concentric rings around the older ones. Over time, the ring of older proteins at the core became smaller and eventually disappeared. Red and green dots were also observed, which the researchers interpreted as vesicles carrying new proteins toward the structure and hauling old proteins away. The researchers note that the “migration” of proteins from the outside to the inside of the structure contradicts the traditional depiction of this process. High-resolution EM studies conducted with ReAsH confirmed that 100- to 150-nm vesicles participate in the delivery of connexins to the cell’s surface. (Science 2002, 296, 503–507) A related paper details FIAsH–cysteine tag binding kinetics and the use of such tags in affinity chromatography, fluorescence anisotropy measurements, and electron microscopy. (J. Am. Chem. Soc. 2002, 124, 6063–6076)

into the protein of interest. Cells expressing this recombinant protein are exposed to the commercially available label FIAsH, a derivative of fluorescein with two arsenics. When the cysteine tag is present, FIAsH binds to it and fluoresces green. Red and blue tags also have been made. The researchers used this approach to study gap junction plaques, which form in the extracellular space between two cells. Each cell provides one or more “hemichannels”—analogous to half a length of pipe—which consist of six connexin proteins arranged around a central pore. When two hemichannels meet, the cells can coordinate their activities by exchanging small signaling molecules, ions, and metabolites. In this study, connexin43 with the cysteine tag was expressed in human cells. Then, FIAsH was added to label the initial connexin43 molecules, and the excess label was washed away. After 4–8 h, the red variant, ReAsH, was added to label newly synthesized con-

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Analytical chemists have long benefited

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the University of North Carolina–Chapel Hill describe the synthesis of a chiral molecu-

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by first synthesizing enantiopure bis(pyridyl)-

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binaphthyl ligands with a >60% overall yield.

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the luminescence is quenched but at signifi-

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rhenium complex to form compounds with

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