Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins
Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins
复制标题
DOI：
10.1529/biophysj.107.120220
复制
发表时间：
2008-06-15
影响因子：
3.4
通讯作者：
Haran, Gilad
中科院分区：
文献类型：
--
作者：
Sherman, Eilon;Itkin, Anna;Haran, Gilad
荧光相关光谱(FCS)是一种灵敏的分析工具，可以在广泛的实验条件下研究生物分子的动力学和流体动力学。目前感兴趣的FCS的一个应用是确定不同状态下的蛋白质分子沿其折叠反应坐标采样的大小，这可以通过测量扩散系数来获得。有人指出，FCS曲线的分析容易产生伪影，从而可能导致错误的尺寸确定。为了为未折叠蛋白质的FCS研究奠定基础，我们首先证明了即使在存在显著改变溶液折射率的高浓度共溶质的情况下，也可以准确地测定小分子以及蛋白质的扩散系数。事实上，研究发现，测量的扩散系数和溶液粘度之间的斯托克斯-爱因斯坦关系即使在高浓度的甘油或盐酸胍(GuHCI)溶液中也成立。这些测量结果构成了研究两种蛋白质变性状态结构的基础，这两种蛋白质是小蛋白L和较大的三结构域蛋白腺苷酸激酶(AK)。FCS被发现对于探测变性状态下展开转变之外的膨胀是有用的。结果表明，蛋白质L的变性状态随变性剂浓度的增加而扩大，这一过程类似于聚合物中从球状到卷曲的转变过程。这一过程至少持续到5M Guhci。另一方面，AK的变性状态似乎不会扩展到2M以上，这一结果与单分子荧光直方图定性一致。由于AK的去折叠转变和卷曲-球状转变都发生在比蛋白质L低得多的变性剂浓度下，因此提出了这两种现象之间的可能联系。
Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCI) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCI. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCI, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.