Naik R R, Kirkpatrick S M, Stone M O
Air Force Research Laboratory, Materials and Manufacturing Directorate, Wright-Patterson Air Force Base, OH 45433-7702, USA.
Biosens Bioelectron. 2001 Dec;16(9-12):1051-7. doi: 10.1016/s0956-5663(01)00226-3.
Coiled-coil proteins are assemblies of two to four alpha-helices that pack together in a parallel or anti-parallel fashion. Coiled-coil structures can confer a variety of functional capabilities, which include enabling proteins, such as myosin, to function in the contractile apparatus of muscle and non-muscle cells. The TlpA protein encoded by the virulence plasmid of Salmonella is an alpha-helical protein that forms an elongated coiled-coil homodimer. A number of studies have clearly established the role of TlpA as a temperature-sensing gene regulator, however the potential use of a TlpA in a thermo-sensor application outside of the organism has not been exploited. In this paper, we demonstrate that TlpA has several characteristics that are common with alpha-helical coiled-coils and its thermal folding and unfolding is reversible and rapid. TlpA is extremely sensitive to changes in temperature. We have also compared the heat-stability of TlpA with other structurally similar proteins. Using a folding reporter, in which TlpA is expressed as a C-terminal fusion with green fluorescent protein (GFP), we were able to use fluorescence as an indicator of folding and unfolding of the fusion protein. Our results on the rapid conformational changes inherent in TlpA support the previous findings and we present here preliminary data on the use of a GFP-TlpA fusion protein as temperature sensor.
卷曲螺旋蛋白是由两到四个α螺旋以平行或反平行方式组装在一起的结构。卷曲螺旋结构可以赋予多种功能能力，包括使肌球蛋白等蛋白质在肌肉和非肌肉细胞的收缩装置中发挥作用。沙门氏菌毒力质粒编码的TlpA蛋白是一种α螺旋蛋白，形成一个细长的卷曲螺旋同源二聚体。许多研究已经明确证实了TlpA作为温度感应基因调节因子的作用，然而，TlpA在生物体之外的热传感器应用中的潜在用途尚未得到开发。在本文中，我们证明TlpA具有一些与α螺旋卷曲螺旋共有的特征，其热折叠和去折叠是可逆且快速的。TlpA对温度变化极为敏感。我们还将TlpA的热稳定性与其他结构相似的蛋白质进行了比较。使用一种折叠报告基因，其中TlpA作为与绿色荧光蛋白（GFP）的C端融合蛋白表达，我们能够将荧光用作融合蛋白折叠和去折叠的指标。我们关于TlpA固有快速构象变化的结果支持了先前的发现，并且我们在此展示了关于使用GFP-TlpA融合蛋白作为温度传感器的初步数据。
