Volumetric characterizations of the native, molten globule and unfolded states of cytochrome c at acidic pH.
Volumetric characterizations of the native, molten globule and unfolded states of cytochrome c at acidic pH.
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酸性 pH 条件下细胞色素 c 的天然熔球和展开状态的体积特征。
DOI：
10.1006/jmbi.1995.0377
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发表时间：
1995
影响因子：
5.6
通讯作者：
K. Breslauer
中科院分区：
文献类型：
--
作者：
T. Chalikian;V. Gindikin;K. Breslauer
细胞色素c可以存在于天然（N），熔融球（MG）或展开（U）状态，这取决于溶液条件。我们已经使用高精度的超声波和密度测定技术来测量体积和可压缩性的变化，伴随着N到MG，N到U和U到MG的蛋白质的过渡。对于N到MG的转变（通过在200 mM CsCl存在下将pH值降低到2诱导），我们测量到体积增加0.014 cm 3 g-1，压缩性增加3.8 x 10（-6）cm 3 g-1 bar-1。对于N到U的转变（通过在不存在盐的情况下将pH值降低到2来诱导），我们测量到体积增加0.010 cm 3 g-1，压缩性降低2.0 x 10（-6）cm 3 g-1 bar-1。对于在pH 2下的U到MG的转变（通过添加CsCl至200 mM诱导），我们测量到0.006 cm 3 g-1的体积增加和6.8 x 10（-6）cm 3 g-1 bar-1的压缩性增加。我们对这些数据进行解释，得出以下关于细胞色素c三种状态的结论。(1)溶剂不可接近的核心被保存在熔融球状态，该核心的体积约为天然细胞色素c固有体积的40%。(2)这个保存下来的熔融球核的绝热压缩系数是61 × 10（-6）bar-1，这个值比天然蛋白质内部的绝热压缩系数高出四倍多。这一结果与保存的MG核的内部是液体状的相一致，而与天然状态的更紧密堆积的固体状内部相反。(3)在细胞色素c的未折叠状态下，只有70 - 80%的完全未折叠构象的表面积暴露于溶剂中，这一结果反映了“变性”状态下的某种程度的有序性。(4)溶剂不可接近的内部的本地，熔融球和展开状态的相对体积波动分别等于0.6%，2.0%和2.9%。这些数据是一致的，与溶剂不可接近的核心的熔融球状态被更松散地包装比核心的天然状态。事实上，熔融球和未折叠状态的波动是如此之高，以至于不能排除形式上掩埋的原子团与溶剂分子短暂接触的可能性。据我们所知，这里报道的数据提供了第一个表征的固有体积和压缩特性的本地，熔融球和未折叠状态的单一蛋白质。我们讨论在目前的蛋白质文献的新的见解，可以从这些数据。
Cytochrome c can exist in a native (N), a molten globule (MG) or an unfolded (U) state depending on solution conditions. We have used high-precision ultrasonic and densimetric techniques to measure volume and compressibility changes accompanying the N to MG, N to U and U to MG transitions of the protein. For the N to MG transition (induced by lowering the pH to 2 in the presence of 200 mM CsCl), we measure a volume increase of 0.014 cm3g-1 and a compressibility increase of 3.8 x 10(-6) cm3g-1bar-1. For the N to U transition (induced by lowering the pH to 2 in the absence of salt), we measure a volume increase of 0.010 cm3 g-1 and a compressibility decrease of 2.0 x 10(-6) cm3 g-1 bar-1. For the U to MG transition at pH 2 (induced by adding CsCl up to 200 mM), we measure a volume increase of 0.006 cm3 g-1 and a compressibility increase of 6.8 x 10(-6) cm3 g-1 bar-1. We interpret these data to reach the following conclusions about the three states of cytochrome c. (1) A solvent-inaccessible core is preserved in the molten globule state, with the volume of this core being about 40% of the intrinsic volume of native cytochrome c. (2) The coefficient of the adiabatic compressibility of this preserved molten globule core is 61 x 10(-6) bar-1, a value that is over four times higher than that of the interior of the native protein. This result is consistent with the interior of the preserved MG core being liquid-like in contrast to the more tightly packed, solid-like interior of the native state. (3) In the unfolded state of cytochrome c, only 70 to 80% of the surface area of a fully unfolded conformation is exposed to the solvent, a result that reflects some level of order in the "denatured" state. (4) The relative volume fluctuations of the solvent-inaccessible interiors of the native, molten globule and unfolded states are equal to 0.6%, 2.0% and 2.9%, respectively. These data are consistent with the solvent-inaccessible core of the molten globule state being much more loosely packed than the core of the native state. In fact, the fluctuations in the molten globule and unfolded states are so high that one cannot exclude the possibility that formally buried atomic groups transiently contact solvent molecules. To the best of our knowledge, the data reported here provide the first characterizations of the intrinsic volume and compressibility properties of the native, molten globule and unfolded states of a single protein. We discuss in terms of the current protein literature the new insights that can be derived from these data.