Membrane Protein Folding and Assembly

 描述(申请人提供)：这里提出的研究是我们实验室多年来对膜蛋白(MPS)折叠和组装进行的生物物理研究的自然延伸，范围从多肽与脂双层的基本相互作用到与von Heijne实验室合作的Sec61易位子将跨膜(TM)螺旋共翻译插入微粒体膜的测量。后一项研究产生了第一个生物疏水性标尺。我们现在正在利用单跨MPS(S-SMPS)来研究TM螺旋在大肠杆菌内膜中的稳定性，以利用沿着两种不同途径插入TM螺旋的可能性：SECA翻译后途径或共翻译信号识别粒子(SRP)途径。我们的全球目标是将TM螺旋稳定性的物理化学原理与跨膜螺旋识别的分子编码定量地联系起来。解决这个问题将为从氨基酸序列预测3D结构奠定必要的基础，这将为研究囊性纤维化等膜蛋白错误折叠障碍提供深入的认识。分子密码用转运子和膜双层之间的Δ-螺旋的表观转移自由能(GAPP)来描述，而物理化学稳定性一般用水和双层之间的转移自由能(ΔGwbi)来描述。ΔGapp决定新生的螺旋是否从转位子进入双层；ΔGwbi决定它是否会留在那里。我们将采取几种方法在ΔGwbi和ΔGapp之间建立更牢固的联系。连接自由能的一种方法是纯粹的生物学方法。我们利用S-SMP CADC与设计的TM片段的嵌合体，发现当信号肽酶切割周质结构域时，一些TM片段从大肠杆菌内膜“脱落”到细胞质中。也就是说，一些TM片段是亚稳定的，仅因为水溶性的周质结构域不能穿过膜而留在膜上。这一显着的现象将使我们能够通过建立一个稳定的阈值，在一个活的生物系统中直接将ΔGAPP与ΔGWBI连接起来。我们在CADC TM节段的亚稳性方面的工作提出了针对S-SMP的根本问题，需要进行研究。我们已经确定CADC需要SecA通过SecYEG插入，但这种插入受SRP的调控。这一效应是一个更大问题的一部分：对于多跨膜蛋白，SECA是运输含有30个或更多氨基酸的周质结构域所必需的。SECA和SRP如何合作实现这一目标完全是未知的。我们假设答案在于对SECA和SRP通路的理解。我们开发了新的方法，(1)保证S-SMPS的SECA单独插入；(2)允许我们在路径选择过程中检查信号序列和TM节段疏水性之间的相互作用。这些想法为我们的四个具体目标提供了理论基础：1.建立TM片段插入后的稳定性阈值(ΔGWBI)和体内SECA驱动的TM插入的生物疏水性标尺(ΔGAPP)。2.使用我们的两种新方法确定途径选择-SRP或SecA-依赖于单个TM片段和信号序列的相对疏水性。3.将分子动力学模拟与分子生物学实验相结合，建立转座子的动力学和静电学之间的联系，这些变化伴随着ΔGAPP的变化，这些变化是由位于Sec61中心的Sec61孔环的大量突变引起的。4.开发物理和计算方法，使用基于用于测定ΔGAPP的TM段的合成TM段，获得ΔGWBI上的直接物理化学数据。

 DESCRIPTION (provided by applicant): The research proposed here is a natural extension of the biophysical studies our lab has carried out over many years on the folding and assembly of membrane proteins (MPs), ranging from fundamental interactions of peptides with lipid bilayers to measurements of the co-translational insertion of transmembrane (TM) helices into microsomal membranes by the Sec61 translocon in collaboration with the von Heijne lab. The latter studies yielded the first biological hydrophobicity scales. We are now engaged in studies of TM helix stability in the inner membranes living E. coli using single-span MPs (S-SMPs) to take advantage of the possibility of inserting TM helices along two different pathways: the SecA post-translational pathway or the co-translational signal recognition particle (SRP) pathway. Our global objective is to connect quantitatively the physicochemical principles of TM helix stability with the molecular code for transmembrane helix recognition by translocons. Solving this problem will lay a necessary foundation for predicting 3D structure from amino acid sequence, which will provide insights into membrane protein misfolding disorders, such as cystic fibrosis. The molecular code is described by the apparent free energy of transfer (ΔGapp) of an -helix between translocon and membrane bilayer whereas physicochemical stability is generally described by the free energy of transfer (ΔGwbi) between water and bilayer. ΔGapp determines whether a nascent helix enters the bilayer from the translocon; ΔGwbi determines whether it will stay there. We will pursue several approaches to establish a firmer connection between ΔGwbi and ΔGapp. One approach to connecting the free energies is purely biological. We have discovered, using chimeras of the S-SMP CadC with designed TM segments, that some TM segments "drop out" of the E. coli inner membrane into the cytoplasm when the periplasmic domain is cleaved by signal peptidase. That is, some TM segments are metastable and remain in the membrane only because the water-soluble periplasmic domain cannot cross the membrane. This remarkable phenomenon will allow us to connect ΔGapp to ΔGwbi directly in a living biological system by establishing a threshold for stability. Our work on the metastability of CadC TM segments has raised fundamental questions about the targeting of S-SMPs that demand investigation. We have determined that CadC requires SecA for insertion via SecYEG, but that insertion is modulated by SRP. This effect is part of a larger problem: For multi-span membrane proteins, SecA is required for the transport of periplasmic domains containing 30 or more amino acids. Just how SecA and SRP cooperate to achieve this is completely unknown. We hypothesize the answer lies in understanding to the SecA and SRP pathways. We have developed new methods that (1) guarantee insertion by SecA alone of S-SMPs and (2) allow us to examine the interplay between signal-sequence and TM-segment hydrophobicities during pathway selection. These ideas provide the rationale for our four specific aims: 1. Establish a post-insertion stability threshold for TM segments (ΔGwbi) and an in vivo a biological hydrophobicity scale for SecA-driven TM insertion (ΔGapp). 2. Determine the dependence of pathway selection-SRP or SecA-on the relative hydrophobicities of single TM segments and signal sequences using our two new methods. 3. Use molecular dynamics simulations in conjunction with molecular biology experiments to establish a connection between the dynamics and electrostatics of translocons that accompany changes in ΔGapp caused by massive mutations of the Sec61 pore ring that lies at the center of Sec61. 4. Develop physical and computational methods for obtaining direct physicochemical data on ΔGwbi using synthetic TM segments based upon TM segments used in determinations of ΔGapp.