We investigated refolding processes of beta-sheeted protein FHA domains (FHA1 domain of Rad53 and Ki67 FHA domain) by cryo-stopped-flow (SF) method combined with far-ultraviolet (far-UV) circular dichroism (CD, the average secondary structure content) and small angle X-ray scattering (SAXS, measuring the radius of gyration). In case of FHA1 domain of Rad53, no detectable time course was observed except the initial burst on its refolding process at 4 degrees C, suggesting that the FHA1 domain of Rad53 was already refolded to its native state within the dead time of the SF apparatus and the rate of the refolding is too fast to be observed at this temperature. In contrast, there was an observable alpha-helical burst at -15 degrees C and -20 degrees C in the presence of 45% ethylene glycol (EGOH) by CD-SF. Besides, the radius of gyration (Rg) of the burst phase intermediate at -20 degrees C shows the intermediate is already compact, and the compaction process was accompanied with the decrease of alpha-helical content at the same temperature. In case of Ki67 FHA domain, ellipticity change at 222 nm was observed on its refolding pathway at -28 degrees C in the presence of 45% EGOH and 2 mM DTT, indicating that Ki67 FHA domain also takes non-native alpha-helix-rich intermediate on its folding pathway. Time-resolved SAXS experiment was done. As the signal/noise ratio is low, we could not observe the time-dependent signal change through the time course. However, the initial Rg value was obtained as 18.2 +/- 0.5 A, which is much smaller than the unfolded Rg value (26.5 +/- 1.2 A), and is slightly larger than the native one (15.9 +/- 1.8 A). These results suggest that Ki67 FHA domain also forms compact non-native alpha-helix-rich intermediate before refolding to its native beta-structure on the refolding pathway. These results are in good agreement with other beta-proteins, such as bovine beta-lactoglobulin (BLG), src SH3 domain proteins. It seems the alpha-helical burst phases appear on the folding pathway of beta-sandwiched proteins.

• PMID: 20466033
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Reference Type

Journal Article

Authors

Matsumura Y, Shinjo M, Mahajan A, Tsai MD, Kihara H

Primary Lit For

RAD53

Additional Lit For

Review For