Golaki Bahram Pooreydy, Aminzadeh Saeed, Karkhane Ali Asghar, Yakhchali Bagher, Farrokh Parisa, Jazii Ferdous Rastgar, Nadimifar Mohammadsadegh
Bioprocess Engineering Group, Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Sharek-e Pajoohesh Km 15, Tehran-Karaj Highway, P.O. Box: 14965/161, Tehran, Iran.
Protein J. 2015 Aug;34(4):267-74. doi: 10.1007/s10930-015-9622-1.
Secondary structure content of proteins in molten globule state is relatively constant while the quantity of tertiary structures clearly declines due to alterations in side-chain packing. In the present study, we analyze the MG state of lipase-3646 for the first time. We introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was found that at pH 3.0 the protein acquires a MG state. Applying far-UV circular dichroism (CD), our analysis on the secondary structure of lipase-3646 revealed a slight change in the MG state intermediate (pH 3.0) compared to the native state (pH 8.5), which this amount of change is common for MG. Measurements in near-UV CD also showed a significant change in the enzyme conformation at pH 3.0 in comparison with the pH 8.5 wherein the protein acquires its native structure. Quenching the fluorescence by applying acrylamide, the amount 23 and 35 M(-1) were measured at pHs 8.5 and 3.0 respectively for stern-volmer constant (KSV). An increase in the enzyme molecular volume in the MG state was confirmed by gel filtration chromatography.
处于熔球态的蛋白质二级结构含量相对恒定，而三级结构的数量则因侧链堆积的改变而明显减少。在本研究中，我们首次分析了脂肪酶-3646的熔球态。我们引入脂肪酶-3646作为研究处于熔球态的蛋白质的性质和行为以及折叠途径的合适模型。应用荧光光谱法，我们测量了脂肪酶-3646在pH值从1.0到12.0范围内的内源荧光和外源荧光。结果发现，在pH 3.0时，该蛋白质呈现熔球态。应用远紫外圆二色光谱（CD），我们对脂肪酶-3646二级结构的分析表明，与天然态（pH 8.5）相比，熔球态中间体（pH 3.0）有轻微变化，这种变化量在熔球态中很常见。近紫外CD测量也显示，与蛋白质获得天然结构的pH 8.5相比，在pH 3.0时酶的构象有显著变化。通过应用丙烯酰胺猝灭荧光，在pH 8.5和3.0时分别测得猝灭常数（KSV）为23和35 M⁻¹。凝胶过滤色谱法证实了处于熔球态的酶分子体积增大。
