Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy
Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy
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DOI：
10.1073/pnas.172524899
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发表时间：
2002-10-29
影响因子：
11.1
通讯作者：
Frieden, C
中科院分区：
文献类型：
--
作者：
Chattopadhyay, K;Saffarian, S;Frieden, C
对肠脂肪酸结合蛋白 (IFABP) 进行了荧光相关光谱 (FCS) 测量，以研究该蛋白质在其天然状态以及 pH 诱导的中间体中的微秒动力学。 IFABP 是一种小型 (15 kDa) 蛋白质，主要由反平行 β 链组成，包围配体结合的大中央空腔。由于该蛋白不含半胱氨酸，因此制备了两个半胱氨酸突变体（Val60Cys 和 Phe62Cys）并用荧光素进行共价修饰。根据荧光测量，其中一种突变体 (Val60Flu) 在蛋白质空腔内具有荧光素部分，而另一种突变体 (Phe62Flu) 中的荧光素暴露于溶剂中。在位置 60 处修饰的蛋白质表明存在约 35 musec 的构象事件，这在其他突变体 (Phe62Flu) 中未见。当蛋白质展开时，这种快速构象事件的幅度在低 pH 条件下急剧下降。测量扩散随 pH 值变化的实验表明，在 pH 值为 3.5 左右时，形成了不同于自然状态的致密状态。稳态荧光和远紫外 CD 表明去折叠发生在 pH 值低于 3 的情况下。
Fluorescence correlation spectroscopy (FCS) measurements have been carried out on the intestinal fatty acid binding protein (IFABP) to study microsecond dynamics of the protein in its native state as well as in pH-induced intermediates. IFABP is a small (15 kDa) protein that consists mostly of antiparallel beta-strands enclosing a large central cavity into which the ligand binds. Because this protein does not contain cysteine, two cysteine mutants (Val60Cys and Phe62Cys) have been prepared and covalently modified with fluorescein. Based on fluorescence measurements, one of the mutants (Val60Flu) has the fluorescein moiety inside the cavity of the protein, whereas the fluorescein is exposed to solvent in the other (Phe62Flu). The protein modified at position 60 demonstrates the presence of a conformational event on the order of 35 musec, which is not seen in the other mutant (Phe62Flu). The amplitude of this fast conformational event decreases sharply at low pH as the protein unfolds. Experiments measuring the diffusion as a function of pH indicate the formation of a compact state distinct from the native state at about pH 3.5. Steady state fluorescence and far-UV CD indicates that unfolding occurs at pH values below pH 3.