Investigating protein folding and unfolding in electrospray nanodrops upon rapid mixing using theta-glass emitters.
Investigating protein folding and unfolding in electrospray nanodrops upon rapid mixing using theta-glass emitters.
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DOI：
10.1021/ac503981c
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发表时间：
2015-01-20
影响因子：
7.4
通讯作者：
Williams, Evan R.
中科院分区：
文献类型：
--
作者：
Mortensen, Daniel N.;Williams, Evan R.
在纳米电喷雾(NanESI)过程中，Theta玻璃发射器被用来快速混合两种溶液，以诱导蛋白质折叠或展开。在纳米ESI过程中，将酸变性肌红蛋白与醋酸铵水溶液混合以提高溶液pH可导致蛋白质折叠。由快速混合实验中折叠构象的相对丰度和折叠时间常数得到反应时间和液滴寿命上限为9±2μS，折叠时间常数为7μS，没有发生血红素再结合，这与该过程较短的液滴寿命和较长的时间常数一致。与酸变性细胞色素c类似的混合实验以及在纳米电沉积过程中产生的折叠表明，根据溶液组成的不同，S的反应时间在7至25μ之间。在与theta-Glass发射器快速混合时，全息肌红蛋白的展开程度比先前报道的要小(Fisher等人。，−)，这一结果归因于小得多的∼1.5μm，平均o.d。这里使用的提示。这些结果表明，在纳米ESI过程中蛋白质折叠或展开的时间框架既取决于初始液滴大小(可以通过改变滴头尖端直径来改变)，也取决于溶液组成。这项研究表明，使用theta-Glass发射器结合质谱学，可以很容易地研究发生在∼10μS时间尺度上的蛋白质折叠或去折叠过程。
Theta-glass emitters are used to rapidly mix two solutions to induce either protein folding or unfolding during nanoelectrospray (nanoESI). Mixing acid-denatured myoglobin with an aqueous ammonium acetate solution to increase solution pH results in protein folding during nanoESI. A reaction time and upper limit to the droplet lifetime of 9 ± 2 μs is obtained from the relative abundance of the folded conformer in these rapid mixing experiments compared to that obtained from solutions at equilibrium and a folding time constant of 7 μs. Heme reincorporation does not occur, consistent with the short droplet lifetime and the much longer time constant for this process. Similar mixing experiments with acid-denatured cytochrome c and the resulting folding during nanoESI indicate a reaction time of between 7 and 25 μs depending on the solution composition. The extent of unfolding of holo-myoglobin upon rapid mixing with theta-glass emitters is less than that reported previously (Fisher et al. , , −), a result that is attributed to the much smaller, ∼1.5 μm, average o.d. tips used here. These results indicate that the time frame during which protein folding or unfolding can occur during nanoESI depends both on the initial droplet size, which can be varied by changing the emitter tip diameter, and on the solution composition. This study demonstrates that protein folding or unfolding processes that occur on the ∼10 μs time scale can be readily investigated using rapid mixing with theta-glass emitters combined with mass spectrometry.
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影响因子：
5.6
作者：
GRIKO, YV;PRIVALOV, PL;KUTYSHENKO, VP
通讯作者：
KUTYSHENKO, VP
DOI：
10.1073/pnas.94.5.1779
发表时间：
1997-03-04
影响因子：
11.1
作者：
Chan, CK;Hu, Y;Hofrichter, J
通讯作者：
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影响因子：
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作者：
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通讯作者：
Williams, Evan R.
DOI：
10.1038/nsb0598-363
发表时间：
1998-05-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Gilmanshin, R;Callender, RH;Dyer, RB
通讯作者：
Dyer, RB
影响因子：
2
作者：
Labowsky, M.
通讯作者：
Labowsky, M.