Temperature-pressure stability of green fluorescent protein: A Fourier transform infrared spectroscopy study
Temperature-pressure stability of green fluorescent protein: A Fourier transform infrared spectroscopy study
复制标题
DOI：
10.1002/bip.10237
复制
发表时间：
2002-11-15
期刊：
影响因子：
2.9
通讯作者：
Vogel, RF
中科院分区：
文献类型：
--
作者：
Scheyhing, CH;Meersman, F;Vogel, RF
绿色荧光蛋白（GFP）作为分子和细胞生物学的标记物被广泛使用。由于其在高压微生物实验中的应用，最近对其在压力下的荧光进行了研究。为了弄清这些变化是否与结构变化有关，我们用傅里叶变换红外光谱法研究了野生型GFP（wtGFP）及其三种红移突变体（AFP、GFP（mut 1）和GFP（mut 2））的压力稳定性。对于野生型GFP、GFP（mut 1）和GFP（mut 2），我们发现高达13-14 kbar的二级结构保持完整，而AFP在10 kbar左右开始解折叠。三维结构负责这种高压稳定性。先前观察到的荧光在低压下的变化是合理的压力引起的弹性效应。高于6 kbar，荧光损失是由于聚集。重新审视GFP的温度稳定性，我们发现一个中间状态沿着wtGFP的解折叠途径被填充沿着。在较高的温度下，解折叠导致wtGFP及其突变体的聚集体的形成。(C)2002 Wiley Periodicals，Inc.
Green fluorescent protein (GFP) is widely used as a marker in molecular and cell biology. For its use in high-pressure microbiology experiments, its fluorescence under pressure was recently investigated. Changes in fluorescence with pressure were found. To find out whether these are related to structural changes, we investigated the pressure stability of wild-type GFP (wtGFP) and three of its red shift mutants (AFP, GFP(mut1) and GFP(mut2)) using Fourier transform infrared spectroscopy. For the wt GFP, GFP(mut1), and GFP(mut2) we found that up to 13-14 kbar the secondary structure remains intact, whereas AFP starts unfolding around 10 kbar. The 3-D structure is held responsible for this high-pressure stability. Previously observed changes in fluorescence at low pressure are rationalized in terms of the pressure-induced elastic effect. Above 6 kbar, loss of fluorescence is due to aggregation. Revisiting the temperature stability of GFP, we found that an intermediate state is populated along the unfolding pathway of wtGFP. At higher temperatures, the unfolding resulted in the formation of aggregates of wtGFP and its mutants. (C) 2002 Wiley Periodicals, Inc.