Conformational Dynamics of Loop 262-274 in G- and F-actin

详细信息    查看全文

• 作者：Alexander Shvetsov ; John D. Stamm ; Martin Phillips ; Dora Warshaviak ; Christian Altenbach ; Peter A. Rubenstein ; Ká ; lmá ; n Hideg ; Wayne L. Hubbell ; and Emil Reisler
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：May 23, 2006
• 年：2006
• 卷：45
• 期：20
• 页码：6541 - 6549
• 全文大小：489K
• 年卷期：v.45,no.20(May 23, 2006)
• ISSN：1520-4995

文摘

According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers onthe opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)₂CA], we showedpreviously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filamentformation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuatingbetween the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)₂CA F-actinon electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphousaggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactionsproceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB(4.4 Å) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with alonger MTS-6 reagent (9.6 Å) did not impair actin polymerization or filament structure. Myosin subfragment1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electronparamagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin couplingand a similar mean interspin distance (~10 Å) in G- and in F-actin, with a broader distance distributionin G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamicswith actin filament formation and stability.