The use of pressure is an advantageous approach to the study of protein structure and dynamics, because it can shift the equilibrium populations of protein conformations toward higher energy states that are not of sufficient population to be observable at atmospheric pressure. Recently, the Hubbell group at the University of California, Los Angeles, reintroduced the application of high pressure to the study of proteins by electron paramagnetic resonance (EPR) spectroscopy. This methodology is possible using X-band EPR spectroscopy due to advances in pressure intensifiers, sample cells, and resonators. In addition to the commercial availability of the pressure generation and sample cells by Pressure Biosciences Inc., a five-loop–four-gap resonator required for the initial high-pressure EPR spectroscopy experiments by the Hubbell group, and those reported here, was designed by James S. Hyde and built and modified at the National Biomedical EPR Center. With these technological advances, we determined the effect of pressure on the essential periplasmic lipopolysaccharide (LPS) transport protein from Escherichia coli, LptA, and one of its binding partners, LptC. LptA unfolds from the N-terminus to the C-terminus, binding of LPS does not appreciably stabilize the protein under pressure, and monomeric LptA unfolds somewhat more readily than oligomeric LptA upon pressurization to 2 kbar. LptC exhibits a fold and relative lack of stability upon LPS binding similar to LptA, yet adopts an altered, likely monomeric, folded conformation under pressure with only its C-terminus unraveling. The pressure-induced changes likely correlate with functional changes associated with binding and transport of LPS.

中文翻译：

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大肠杆菌脂多糖转运蛋白 LptA 和 LptC 的高压 EPR 光谱研究

使用压力是研究蛋白质结构和动力学的一种有利方法，因为它可以将蛋白质构象的平衡种群转移到更高的能量状态，而在大气压下，这种状态的种群数量不足以观察到。最近，加州大学洛杉矶分校的哈贝尔小组重新引入了高压在电子顺磁共振（EPR）光谱研究中的应用。由于压力增强器、样品池和谐振器的进步，这种方法可以使用 X 波段 EPR 光谱。除了压力生物科学公司的压力产生和样品池的商业可用性之外，Hubbell 小组最初的高压 EPR 光谱实验所需的五回路四间隙谐振器，和这里报道的那些，是由 James S. Hyde 设计的，并在国家生物医学 EPR 中心建造和修改。随着这些技术进步，我们确定了压力对来自大肠杆菌的必需周质脂多糖 (LPS) 转运蛋白 LptA 及其结合伙伴之一 LptC 的影响。LptA 从 N 端向 C 端展开，LPS 的结合在压力下不会明显稳定蛋白质，并且单体 LptA 在加压至 2 kbar 时比寡聚 LptA 更容易展开。LptC 在 LPS 结合时表现出折叠和相对缺乏稳定性，类似于 LptA，但在压力下采用改变的、可能是单体的折叠构象，只有其 C 端解开。