Efficacy of macromolecular crowding in forcing proteins to fold
Efficacy of macromolecular crowding in forcing proteins to fold
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DOI：
10.1016/s0301-4622(02)00148-5
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发表时间：
2002-12-10
影响因子：
3.8
通讯作者：
Bolen, DW
中科院分区：
文献类型：
--
作者：
Qu, YX;Bolen, DW
用本质上非结构的蛋白质，还原和羧胺RNaseT1(TCAM)来确定大分子排泄剂增加折叠平衡常数的程度。TCAM在水相体系中不具有催化活性，但在添加400 mg/ml的葡聚糖70后具有催化活性。观察到的活度约占溶液中可用TCAM总量的16%。我们将这一结果解释为，在400 mg/ml的葡聚糖70存在下，16%的TCAM成为折叠蛋白质，这意味着折叠的平衡常数大约增加了五倍。在0、100、200和300 mg/ml葡聚糖70存在下进行肌氨酸诱导的TCAM折叠，由线性外推法测定的表观DeltaG(N-D)(O)值给出在零肌氨酸浓度和400 mg/ml葡聚糖70存在下形成的TCAM折叠的估计22%。用该方法测定TCAM折叠平衡常数约为7.5倍。总体而言，结果表明，大分子挤压剂在促进这种固有的非结构蛋白质的折叠方面只有一定的效果。(C)2002 Elsevier Science B.V.保留所有权利。
The intrinsically unstructured protein, reduced and carboxyamidated RNase T1 (TCAM) was used to determine the degree to which macromolecular crowding agents increase the equilibrium constant for folding. TCAM is not catalytically active in an aqueous assay system alone, but becomes catalytically active on addition of 400 mg/ml dextran 70. The activity observed accounts for approximately 16% of the total available TCAM in solution. We interpret this result to mean that 16% of the TCAM becomes folded protein in the presence of the 400 mg/ml dextran 70, and this translates into an approximately five-fold increase in the equilibrium constant for folding. Sarcosine-induced folding of TCAM was performed in the presence of 0, 100, 200 and 300 mg/ml dextran 70, and apparent DeltaG(N-D)(o), values determined from the linear extrapolation method provide an estimated 22% folded TCAM formed in the limit of zero sarcosine concentration and in presence of 400 mg/ml dextran 70. The increase in TCAM folding equilibrium constant using this method of determination is approximately 7.5-fold. Overall, the results indicate that macromolecular crowding agents are only modestly effective in promoting folding of this intrinsically unstructured protein. (C) 2002 Elsevier Science B.V. All rights reserved.