细胞色素c在酸性pH值下天然态、熔球态和去折叠态的体积表征。

Volumetric characterizations of the native, molten globule and unfolded states of cytochrome c at acidic pH.

作者信息

Chalikian T V, Gindikin V S, Breslauer K J

机构信息

Department of Chemistry Rutgers, State University of New Jersey, Piscataway 08855, USA.

出版信息

J Mol Biol. 1995 Jul 7;250(2):291-306. doi: 10.1006/jmbi.1995.0377.

DOI:10.1006/jmbi.1995.0377

PMID:7608975

Abstract

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.

摘要

细胞色素c可根据溶液条件以天然态（N）、熔球态（MG）或未折叠态（U）存在。我们使用高精度超声和密度测量技术来测量蛋白质从N态到MG态、N态到U态以及U态到MG态转变过程中伴随的体积和压缩性变化。对于N到MG的转变（在200 mM CsCl存在下将pH值降至2诱导），我们测得体积增加0.014 cm³g⁻¹，压缩性增加3.8×10⁻⁶ cm³g⁻¹bar⁻¹。对于N到U的转变（在无盐情况下将pH值降至2诱导），我们测得体积增加0.010 cm³ g⁻¹，压缩性降低2.0×10⁻⁶ cm³ g⁻¹ bar⁻¹。对于pH值为2时U到MG的转变（通过添加高达200 mM的CsCl诱导），我们测得体积增加0.006 cm³ g⁻¹，压缩性增加6.8×10⁻⁶ cm³ g⁻¹ bar⁻¹。我们对这些数据进行解读，得出关于细胞色素c三种状态的以下结论。（1）在熔球态中保留了一个溶剂不可及的核心，该核心的体积约为天然细胞色素c固有体积的40%。（2）这个保留的熔球态核心的绝热压缩系数为61×10⁻⁶ bar⁻¹，该值比天然蛋白质内部的系数高出四倍多。这一结果与保留的MG核心内部呈液态相一致，与天然态更紧密堆积、固态的内部形成对比。（3）在细胞色素c的未折叠态中，完全未折叠构象的表面积只有70%至80%暴露于溶剂中，这一结果反映了“变性”态中存在一定程度的有序性。（4）天然态、熔球态和未折叠态的溶剂不可及内部的相对体积波动分别为0.6%、2.0%和2.9%。这些数据与熔球态的溶剂不可及核心比天然态的核心堆积松散得多相一致。事实上，熔球态和未折叠态的波动非常大，以至于不能排除原本埋藏的原子基团与溶剂分子短暂接触的可能性。据我们所知，这里报道的数据首次对单一蛋白质的天然态、熔球态和未折叠态的固有体积和压缩性特性进行了表征。我们根据当前的蛋白质文献讨论了从这些数据中可以得出的新见解。