Kleinschmidt J H, Tamm L K
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville 22906-0011, USA.
Biochemistry. 1996 Oct 8;35(40):12993-3000. doi: 10.1021/bi961478b.
The mechanism of folding and membrane insertion of integral membrane proteins, including helix bundle and beta-barrel proteins is not well understood. A key question is whether folding and insertion are coupled or separable processes. We have used the beta-barrel outer membrane protein A (OmpA) of Escherichia coli as a model to study the kinetics of folding and insertion into dioleoylphosphatidylcholine (DOPC) bilayers, as a function of temperature by gel electrophoresis, protease digestion, and fluorescence spectroscopy. OmpA was unfolded in 8 M urea solution (without detergent), and refolding and membrane insertion was initiated by rapid dilution of the urea concentration in the presence of phospholipid vesicles. In addition to the kinetically unresolved hydrophobic collapse in water, the time course of refolding of OmpA into DOPC bilayers exhibited three kinetic phases over a large temperature range. The first step was fast (k1 = 0.16 min-1) and not very dependent on temperature. The second step was up to two orders of magnitude slower at low temperatures (2 degrees C), but approached the rate of the first step at higher temperatures (40 degrees C). The activation energy for this process was 46 +/- 4 kJ/mol. A third slow process (k3 = 0.9 x 10(-2) min-1 at 40 degrees C) was observed at the higher temperatures. These results suggest that at least two membrane-bound intermediates exist when OmpA folds and inserts into lipid bilayers. We also show that both membrane-bound intermediates can be stabilized in fluid lipid bilayers at low temperatures. These intermediates share many properties with the adsorbed/partially inserted form of OmpA that was previously characterized in gel phase lipid bilayers [Rodionova et al. (1995) Biochemistry 34, 1921-1929]. Temperature jump experiments demonstrate, that the low-temperature intermediates can be rapidly converted to fully inserted native OmpA. On the basis of these and previous results, we present a simple folding model for beta-barrel membrane proteins, in which folding and membrane insertion are coupled processes which involve at least four kinetically distinguishable steps.
包括螺旋束蛋白和β桶蛋白在内的整合膜蛋白的折叠及插入膜的机制尚未完全明确。一个关键问题是折叠和插入过程是相互耦合的还是可分离的。我们以大肠杆菌的β桶外膜蛋白A（OmpA）为模型，通过凝胶电泳、蛋白酶消化和荧光光谱法研究其在不同温度下折叠并插入二油酰磷脂酰胆碱（DOPC）双层膜的动力学过程。OmpA在8 M尿素溶液（无去污剂）中展开，在磷脂囊泡存在的情况下，通过快速稀释尿素浓度启动重折叠和膜插入过程。除了在水中动力学上无法解析的疏水塌缩外，OmpA重折叠进入DOPC双层膜的时间进程在较大温度范围内呈现出三个动力学阶段。第一步很快（k1 = 0.16 min-1），且对温度不太敏感。第二步在低温（2℃）时慢达两个数量级，但在较高温度（40℃）时接近第一步的速率。该过程的活化能为46±4 kJ/mol。在较高温度下观察到第三个缓慢过程（40℃时k3 = 0.9×10-2 min-1）。这些结果表明，OmpA折叠并插入脂质双层膜时至少存在两种膜结合中间体。我们还表明，两种膜结合中间体在低温下的流体脂质双层膜中均可稳定存在。这些中间体与先前在凝胶相脂质双层膜中表征的OmpA的吸附/部分插入形式具有许多共同特性[罗季奥诺娃等人（1995年）《生物化学》34卷，1921 - 1929页]。温度跳跃实验表明，低温中间体可迅速转化为完全插入的天然OmpA。基于这些及先前的结果，我们提出了一个简单的β桶膜蛋白折叠模型，其中折叠和膜插入是相互耦合的过程，涉及至少四个动力学上可区分的步骤。
