Hexaporins: the rational design of transmembrane channels

我们的研究涉及了解生物学如何使用分子构建块构建功能结构，如核酸（DNA和RNA）糖，蛋白质和脂质。后两个是这项拨款申请的主题。蛋白质分子是氨基酸的聚合物，可以折叠成确定的三维（3D）功能结构。例如，胶原蛋白为我们的大部分组织提供支架;血红蛋白将氧气从肺部输送到活动器官;己糖激酶分解含葡萄糖的食物以帮助提供生物能量。基本上，蛋白质中有两种类型的氨基酸：疏水性的，字面上是“讨厌水”，和极性的，可溶于水。一个既有极性又有疏水性的水溶性蛋白质会折叠，使其大部分极性氨基酸在表面与水接触，并将大部分疏水性氨基酸埋在里面。然而，大部分生物学是在细胞膜之间或细胞膜内部的界面上进行的，这些界面并不是简单的充满水的空间，而且还需要一组不同的蛋白质。细胞周围的生物膜主要由脂质分子组成。脂质也有两个不同的疏水和极性区域。在膜中，许多脂质聚集在一起形成双层，其中一片脂质与另一片脂质相互作用，将疏水部分掩埋，使极性部分暴露于水;就像三明治中的面包（在这个类比中是极性部分）在外面，而馅料（疏水部分）在中间。这种组织在很大程度上形成了不可渗透的屏障，这在生物学中提出了一个问题，并且需要其他分子，即跨膜蛋白，来促进跨膜的运输和通信。大自然利用这些蛋白质执行许多功能，例如允许营养物质进入细胞;排泄废物;输出防御分子;跨膜传递信号;甚至将光转化为化学能。跨膜蛋白质具有与水溶性蛋白质不同的整体化学性质;它们在外部和内部都是疏水性的。最近，我们发现了一种新型的水溶性蛋白质结构，我们称之为CC-Hex。它有6条蛋白质链，每条蛋白质链都折叠成螺旋状。这些束形成一个圆柱体，有一个洞穿过它，有点像一堆波罗薄荷糖。这种结构类似于称为通道的跨膜蛋白。在这里，我们建议通过合理的蛋白质设计将水溶性CC-Hex转化为跨膜蛋白。关键是我们了解CC-Hex的化学和结构，这将指导我们的设计。为什么这样做？著名物理学家理查德·费曼（Richard Feynman）曾说过，他无法建造的东西，他不理解。这是我们采用的原则：我们将研究天然的跨膜蛋白，从中学习，然后通过设计CC-Hex的简化跨膜通道来测试我们的理解。这可能有失败的风险，但潜在的回报是很高的：我们至少可以了解生物学的一些组成部分是如何组装的;也许我们可以应用这种理解来创造新的蛋白质，这些蛋白质可能会在基础科学和生物技术的其他领域得到应用。例如，一类称为水通道蛋白的天然跨膜通道运输和控制水穿过细胞膜的平衡。例如，在肾脏中，水通道蛋白从尿液中回收水，将其浓缩以帮助避免脱水。水通道蛋白是一种复杂的大分子。如果我们能够在像CC-Hex这样的小蛋白质中捕获它们的特性，我们就有可能生产出在水净化和脱盐设备中具有潜在应用的新分子。

Our research is concerned with understanding how biology builds functional structures using molecular building blocks, such as nucleic acids (DNA and RNA) sugars, proteins and lipids. The latter two are the subjects of this grant proposal.Protein molecules are polymers of amino acids that fold into defined three-dimensional (3D) functional structures. For example, collagen provides scaffolding in most of our tissues; haemoglobin transports oxygen from the lungs to active organs; and hexokinase breaks down glucose-containing foodstuffs to help provide energy in biology.Many proteins fold and function in water. Essentially, there are two types of amino acid in proteins: hydrophobic ones, which are literally "water hating", and polar ones, which are soluble in water. A water-soluble protein with both polar and hydrophobic parts will fold to put most of its polar amino acids on its surface and in contact with water, and bury most of its hydrophobic amino acids.However, much of biology goes on at the interfaces between, or within the membranes of cells, and these are not simple water-filled spaces, and a different set of proteins is needed.Biological membranes surrounding cells are largely made up of lipid molecules. Lipids also have two distinct hydrophobic and polar regions. In membranes, many lipids aggregate together to form a bilayer, in which one leaf of lipids interacts with another burying the hydrophobic parts, leaving the polar parts exposed to water; much like in a sandwich with the bread (the polar parts in this analogy) on the outside, and the filling (the hydrophobic parts) in the middle. This organisation makes largely impermeable barriers, which presents a problem in biology, and other molecules, namely membrane-spanning proteins, are needed to facilitate transport and communication across the membrane. Nature uses these proteins to perform many functions, such as allowing nutrients into cells; excreting waste; exporting defence molecules; conveying signals across membranes; and even converting light into chemical energy.Membrane-spanning proteins have a different overall chemistry to water-soluble proteins; they are hydrophobic on both the outside and the inside. This makes them more difficult to study, and harder to understand.Recently, we discovered a new type of water-soluble protein structure, which we call CC-Hex. It has 6 protein chains, each of which folds up into a helix. These bundle to form a cylinder with a hole through it, a little like a stack of polo mints. This structure resembles membrane-spanning proteins called channels. Here, we propose to turn the water-soluble CC-Hex into a membrane-spanning protein by rational protein design. The key is that we understand both the chemistry and the structure of CC-Hex, which will guide our designs.Why do this? The famous physicist Richard Feynman remarked that what he could not build, he did not understand. This is the principle that we have adopted: we will look at natural membrane-spanning proteins, learn from them, and then test our understanding by designing simplified membrane-spanning channels from CC-Hex. There is a risk that this might not work, but the potential rewards are high: we stand to learn how some of biology's components assemble at the very least; and possibly we could apply this understanding to create new proteins that might find applications in other areas of fundamental science and biotechnology.For instance, a class of natural membrane-spanning channels known as the aquaporins transport and control the balance of water across cell membranes. As an example, in the kidneys aquaporins recover water from urine concentrating it to help avoid dehydration. Aquaporins are large complicated molecules. If we could capture their properties in a small protein like CC-Hex, we could possibly produce new molecules with potential application in water-purification and desalination devices.