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首页> 外文期刊>Biochemistry >MOTIONAL DYNAMICS OF A BURIED TRYPTOPHAN REVEALS THE PRESENCE OF PARTIALLY STRUCTURED FORMS DURING DENATURATION OF BARSTAR
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MOTIONAL DYNAMICS OF A BURIED TRYPTOPHAN REVEALS THE PRESENCE OF PARTIALLY STRUCTURED FORMS DURING DENATURATION OF BARSTAR

机译：隐秘的TRYPTOPHAN的运动动力学揭示了BARSTAR脱模过程中存在部分结构化的形式

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A double mutant of the single-domain protein barstar having a single tryptophan (W53) was made by mutating the remaining two tryptophans (W38 and W44) into phenylalanines. W53 is buried in the core of barstar. Time-resolved fluorescence of the mutant barstar (W38FW44F) showed that W53 has a single fluorescence lifetime in the native (N) state and has three lifetimes in the molten globule-like low-pH (A) form. Quenching of fluorescence by either KI or acrylamide showed that W53 is solvent inaccessible in the N-state and fairly accessible in the A-form. The denaturation of W38FW44F by guanidine hydrochloride (GdnHCl) was monitored by several probes: near-UV and far-UV circular dichroism (CD), fluorescence intensity, and steady-state and time-resolved fluorescence anisotropy. While the unfolding transitions observed through CD and fluorescence intensity coincided with each other (midpoint approximate to 1.8 M GdnHCl), the transition observed through the steady-state fluorescence anisotropy was markedly different from others. Initially, the anisotropy increased with the increase in the concentration of GdnHCl and decreased subsequently. The midpoint of this titration was 2.2 M GdnHCl. Picosecond time-resolved fluorescence anisotropy showed that W38FW44F has a single rotational correlation time of 4.1 ns in the native (N) state and 1.5 ns in the unfolded (U) state (6 M GdnHCl). These could be explained as being due to the absence of motional freedom of W53 in the N-state and the presence of rotational freedom in the U-state. In the intermediate concentration region (1.8-3.0 M GdnHCl), the anisotropy decays showed at least two correlation times, similar to 1 and 6-12 ns, These two correlation times are ascribed to partially structured forms leading to hindered rotation of W53. Thus, the usefulness of time-resolved fluorescence anisotropy in detecting partially folded structures is demonstrated.
机译：通过将剩余的两个色氨酸（W38和W44）突变为苯丙氨酸，可制成具有单个色氨酸（W53）的单域蛋白barstar的双突变体。 W53埋在barstar的核心中。突变型barstar（W38FW44F）的时间分辨荧光表明，W53在天然（N）状态下具有单个荧光寿命，而在熔融小球状低pH（A）形式下具有三个寿命。 KI或丙烯酰胺对荧光的猝灭表明，W53在N态下是溶剂不可及的，而在A形式下是相当可及的。用几种探针监测W38FW44F被盐酸胍（GdnHCl）的变性：近紫外和远紫外圆二色性（CD），荧光强度以及稳态和时间分辨荧光各向异性。通过CD观察到的展开转变与荧光强度一致（中点约为1.8 M GdnHCl），而通过稳态荧光各向异性观察到的转变则明显不同。最初，各向异性随着GdnHCl浓度的增加而增加，随后降低。该滴定的中点是2.2M GdnHCl。皮秒时间分辨的荧光各向异性表明，W38FW44F在自然（N）状态下具有4.1 ns的单个旋转相关​​时间，在未折叠（U）状态下（6 M GdnHCl）具有1.5 ns的单个旋转相关​​时间。可以将这些解释为归因于N状态下W53的运动自由度不存在以及U状态下存在旋转自由度。在中等浓度区域（1.8-3.0 M GdnHCl），各向异性衰减显示至少两个相关时间，类似于1和6-12 ns。这两个相关时间归因于部分结构化形式，导致W53旋转受阻。因此，证明了时间分辨荧光各向异性在检测部分折叠结构中的有用性。

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