Fluorescence probing of yeast actin subdomain 3/4 hydrophobic loop 262-274 - Actin-actin and actin-myosin interactions in actin filaments
Fluorescence probing of yeast actin subdomain 3/4 hydrophobic loop 262-274 - Actin-actin and actin-myosin interactions in actin filaments
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DOI：
10.1074/jbc.272.27.16829
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发表时间：
1997-07-04
影响因子：
4.8
通讯作者：
Rubenstein, PA
中科院分区：
文献类型：
--
作者：
Feng, L;Kim, E;Rubenstein, PA
第262-274位残基在肌动蛋白亚结构域3和4之间形成一个环，该环可能在肌动蛋白丝的形成和稳定中起重要作用。为了直接评估该环的行为，我们将酵母肌动蛋白的Ser（265）突变为半胱氨酸（S265 C），并创建了另一个突变体通过将S265 C肌动蛋白的Cys（374）改变为丙氨酸，这些改变允许我们将芘马来酰亚胺化学计量地连接到Cys（374）或Cys（265）上，这些突变对蛋白酶敏感性、固有ATP酶活性、以及标记或未标记的G-肌动蛋白的热稳定性。标记或未标记的环状半胱氨酸的存在不影响肌动蛋白激活的S1 ATP酶活性或肌动蛋白的体外运动性。标记或未标记的两种突变体肌动蛋白都比野生型（WT）肌动蛋白更快地形成有核丝，尽管临界浓度不受影响。环（S265 C/C374 A）探针的荧光强度降低，双标记肌动蛋白（S265 C）的荧光强度比单个荧光团的荧光强度之和低约50%。在标记的WT和S265 C/C374 A肌动蛋白的共聚物中也观察到猝灭。在标记的S265 C F-肌动蛋白和标记的S265 C/C374 A-WT肌动蛋白共聚物的发射光谱中存在激基缔合物峰。这些结果表明，在细丝中，相当大一部分单体的C-末端芘直接与相邻单体的环芘相互作用，使两个半胱氨酸硫彼此在18埃内。最后，当与标记的S265 C/C374 A F-肌动蛋白结合时，肌球蛋白S1而不是原肌球蛋白引起环探针荧光的增加，这两种蛋白质对激基缔合物荧光没有影响。这些结果有助于建立F-肌动蛋白单体的取向，并表明S1与肌动蛋白亚结构域1和2的结合影响亚结构域3和4之间的环的环境。
Residues 262-274 form a loop between subdomains 3 and 4 of actin, This loop may play an important role in actin filament formation and stabilization, To assess directly the behavior of this loop, we mutated Ser(265) Of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys(374) Of S265C actin to alanine, These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys(374) Or Cys(265), These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin, Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected, Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was similar to 50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins, An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 Angstrom of one another, Finally, when bound to labeled S265C/C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe, Both proteins had no effect on excimer fluorescence, These results help establish the orientation of monomers in F-actin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.