Bobkov Andrey A, Muhlrad Andras, Kokabi Kaveh, Vorobiev Sergey, Almo Steven C, Reisler Emil
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
J Mol Biol. 2002 Nov 1;323(4):739-50. doi: 10.1016/s0022-2836(02)01008-2.
Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)<or=0.05 microM for both CaATP-G-actin and F-actin. Cofilin binding enhanced the fluorescence of dansyl ethylenediamine (DED) attached to Gln41 on the DNase I binding loop of skeletal muscle F-actin and decreased the fluorescence of AEDANS at Cys41 on yeast Q41C/C374S mutant F-actin. However, cofilin had no effect on the spectral properties of DED or AEDANS on CaATP-G-actin. Fluorescence energy transfer (FRET) from tryptophan residues to DED at Gln41 on skeletal muscle actin and to AEDANS at Cys41 on yeast Q41C/C374S actin was decreased by cofilin binding to F- but not to G-actin. Cofilin inhibited strongly the rate of interprotomer disulfide cross-linking of Cys41 to Cys374 on yeast Q41C mutant F-actin. Binding of cofilin enhanced excimer formation between pyrene probes attached to Cys41 and Cys374 on Q41C F-actin. These results indicate that cofilin alters the interface between subdomains 1 and 2 and shifts the DNase I binding loop away from subdomain 1 of an adjacent actin protomer. Cofilin reduced FRET from tryptophan residues to 4-azido-2-nitrophenyl-putrescine (ANP) at Gln41 in skeletal muscle F-but not in G-actin. However, following the interprotomer cross-linking of Gln41 to Cys374 in F-actin by ANP, cofilin binding did not change FRET from the tryptophan residues to ANP. This suggests that cofilin binding and the conformational effect on F-actin are not coupled tightly. Overall, this study provides solution evidence for the weakening of longitudinal, subdomain 2/1 contacts in F-actin by cofilin.
在溶液中研究了酵母丝切蛋白对骨骼肌肌动蛋白和酵母肌动蛋白的结构影响。丝切蛋白与天然肌动蛋白的结合是非协同性的，在1:1摩尔比时达到饱和，对于CaATP-G-肌动蛋白和F-肌动蛋白，解离常数K(d)≤0.05μM。丝切蛋白的结合增强了附着在骨骼肌F-肌动蛋白DNase I结合环上Gln41处的丹磺酰乙二胺（DED）的荧光，并降低了酵母Q41C/C374S突变体F-肌动蛋白Cys41处的5-(2-氨基乙基)氨基萘-1-磺酸（AEDANS）的荧光。然而，丝切蛋白对CaATP-G-肌动蛋白上DED或AEDANS的光谱特性没有影响。丝切蛋白与F-肌动蛋白而非G-肌动蛋白结合后，骨骼肌肌动蛋白上色氨酸残基到Gln41处DED以及酵母DED以及酵母Q41C/C374S肌动蛋白上Cys41处AEDANS的荧光能量转移（FRET）降低。丝切蛋白强烈抑制酵母Q41C突变体F-肌动蛋白上Cys41与Cys374之间的原聚体间二硫键交联速率。丝切蛋白的结合增强了附着在Q41C F-肌动蛋白上Cys41和Cys374处的芘探针之间的准分子形成。这些结果表明，丝切蛋白改变了亚结构域1和2之间的界面，并使DNase I结合环远离相邻肌动蛋白原聚体的亚结构域1。丝切蛋白降低了骨骼肌F-肌动蛋白而非G-肌动蛋白上色氨酸残基到Gln41处的4-叠氮基-2-硝基苯基-腐胺（ANP）的FRET。然而，在通过ANP使F-肌动蛋白中Gln41与Cys374进行原聚体交联后，丝切蛋白的结合并未改变色氨酸残基到ANP的FRET。这表明丝切蛋白的结合与对F-肌动蛋白的构象效应没有紧密耦合。总体而言，本研究为丝切蛋白削弱F-肌动蛋白中亚结构域2/1的纵向接触提供了溶液证据。
