T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Biophys Chem. 2011 Nov;159(1):194-204. doi: 10.1016/j.bpc.2011.06.013. Epub 2011 Jul 6.
OmpA is one of only a few transmembrane proteins whose folding and stability have been investigated in detail. However, only half of the OmpA mass encodes its transmembrane β-barrel; the remaining sequence is a soluble domain that is localized to the periplasmic side of the outer membrane. To understand how the OmpA periplasmic domain contributes to the stability and folding of the full-length OmpA protein, we cloned, expressed, purified and studied the OmpA periplasmic domain independently of the OmpA transmembrane β-barrel region. Our experiments showed that the OmpA periplasmic domain exists as an independent folding unit with a free energy of folding equal to -6.2 (±0.1) kcal mol(-1) at 25°C. Using circular dichroism, we determined that the OmpA periplasmic domain adopts a mixed alpha/beta secondary structure, a conformation that has previously been used to describe the partially folded non-native state of the full-length OmpA. We further discovered that the OmpA periplasmic domain reduces the self-association propensity of the unfolded barrel domain, but only when covalently attached (in cis). In vitro folding experiments showed that self-association competes with β-barrel folding when allowed to occur before the addition of membranes, and the periplasmic domain enhances the folding efficiency of the full-length protein by reducing its self-association. These results identify a novel chaperone function for the periplasmic domain of OmpA that may be relevant for folding in vivo. We have also extensively investigated the properties of the self-association reaction of unfolded OmpA and found that the transmembrane region must form a critical nucleus comprised of three molecules before undergoing further oligomerization to form large molecular weight species. Finally, we studied the conformation of the unfolded OmpA monomer and found that the folding-competent form of the transmembrane region adopts an expanded conformation, which is in contrast to previous studies that have suggested a collapsed unfolded state.
OmpA 是少数几种其折叠和稳定性已被详细研究的跨膜蛋白之一。然而，只有 OmpA 质量的一半编码其跨膜 β-桶；其余序列是一个可溶性结构域，位于外膜的周质侧。为了了解 OmpA 周质结构域如何有助于全长 OmpA 蛋白的稳定性和折叠，我们独立于 OmpA 跨膜 β-桶区域克隆、表达、纯化和研究了 OmpA 周质结构域。我们的实验表明，OmpA 周质结构域作为一个独立的折叠单元存在，其折叠自由能在 25°C 时等于-6.2（±0.1）千卡摩尔（-1）。使用圆二色性，我们确定 OmpA 周质结构域采用混合的 α/β二级结构，这种构象以前曾用于描述全长 OmpA 的部分折叠非天然状态。我们进一步发现，OmpA 周质结构域降低了未折叠桶结构域的自缔合倾向，但只有在共价连接（顺式）时才会发生。体外折叠实验表明，当允许在添加膜之前发生时，自缔合会与 β-桶折叠竞争，并且周质结构域通过降低其自缔合来提高全长蛋白的折叠效率。这些结果确定了 OmpA 周质结构域的一种新的伴侣功能，这可能与体内折叠有关。我们还广泛研究了未折叠 OmpA 自缔合反应的性质，并发现跨膜区域必须形成由三个分子组成的临界核，然后才能进一步寡聚形成大分子量物种。最后，我们研究了未折叠 OmpA 单体的构象，发现折叠 competent 形式的跨膜区域采用扩展构象，这与先前研究表明的折叠状态相反。
