Andrec M, Hill R B, Prestegard J H
Department of Chemistry, Yale University, New Haven, Connecticut 06511, USA.
Protein Sci. 1995 May;4(5):983-93. doi: 10.1002/pro.5560040518.
The acyl carrier protein (ACP) of Escherichia coli is a 77-amino acid, highly negatively charged three-helix protein that plays a central role in fatty acid biosynthesis. Previous NMR studies have suggested the presence of multiple conformations and marginally stable secondary structural elements. The stability of these elements is now examined by monitoring amide exchange in apo-ACP using NMR-based methods. Because ACP exhibits many rapid exchange rates, application of traditional isotope exchange methods is difficult. In one approach, heteronuclear correlation experiments with pulsed field-gradient coherence selection have reduced the time needed to collect two-dimensional 1H-15N correlation spectra to the point where measurement of exchange of amide protons for deuterium on the timescale of minutes can be made. In another approach, water proton selective inversion-exchange experiments were performed to estimate the exchange rates of protons exchanging on timescales of less than a second. Backbone amide protons in the region of helix II were found to exchange significantly more rapidly than those in helices I and III, consistent with earlier structural models suggesting a dynamic disruption of the second helix. Highly protected amides occur on faces of the helices that may pack into a hydrophobic core present in a partially disrupted state.
大肠杆菌的酰基载体蛋白（ACP）是一种由77个氨基酸组成的、带高度负电荷的三螺旋蛋白，在脂肪酸生物合成中起核心作用。先前的核磁共振研究表明存在多种构象以及略微稳定的二级结构元件。现在通过使用基于核磁共振的方法监测脱辅基ACP中的酰胺交换来检测这些元件的稳定性。由于ACP表现出许多快速交换速率，传统同位素交换方法的应用很困难。在一种方法中，采用脉冲场梯度相干选择的异核相关实验将收集二维1H-15N相关谱所需的时间缩短到了可以在几分钟的时间尺度上测量酰胺质子与氘交换的程度。在另一种方法中，进行了水质子选择性反转交换实验，以估计在小于一秒的时间尺度上交换的质子的交换速率。发现螺旋II区域中的主链酰胺质子比螺旋I和III中的主链酰胺质子交换得明显更快，这与早期的结构模型一致，该模型表明第二个螺旋存在动态破坏。高度受保护的酰胺出现在螺旋面上，这些螺旋面可能堆积成处于部分破坏状态的疏水核心。
