( bound to tubulin molecules. If GDP some...)The making and breaking of microtubules

bound to tubulin molecules. If GDP somehow causes protofilaments to curl up tightly, GTP does the opposite, causing protofilaments to straighten and assemble themselves into microtubules. But because GTP can rapidly hydrolyze into GDP, capturing transitional states of GTP-bound tubulin directly was impractical.

Instead the researchers worked with a similar molecule, GMPCPP, an analogue of GTP that binds to the same site but is not vulnerable to hydrolysis. Using cryo-EM they were able to capture GMPCPP-bound tubulin during the initial steps of microtubule growth.

At low temperatures, GMPCPP-bound tubulin forms curved ribbons of a few protofilaments side by side; as they grow wider the ribbons close into very large helical tubes with a diameter of 500 angstroms, instead of the slim, 25-angstrom diameter of a microtubule (an angstrom is a ten-billionth of a meter, roughly the dimension of a small atom).

Raised to body temperature, however, the protofilamentary ribbons immediately straighten and convert to normal microtubules, suggesting that the ribbons and gently curved sheets correspond to the polymerizing protofilaments at the end of a growing microtubule.

Making contacts:

In 1998, when she was a postdoctoral fellow in the laboratory of Kenneth Downing, a senior scientist in Berkeley Lab's Life Sciences Division, Nogales obtained the atomic structure of tubulin in its stable, straight form, using electron crystallography methods, in which two-dimensional arrays of tubulin "flat" polymers were studied using cryo-EM and electron diffraction.

By "docking" this crystallographic model within the structures they have now obtained for the polymerization intermediates at more modest resolution, Nogales and Wang have been able to see changes in tubulin molecules, just how their capacity to interact with other molecules is altered by binding to GTP or GDP, and how these alterations control bending and flexibility.

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Source:DOE/Lawrence Berkeley National Laboratory

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