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来构建功能结构，例如核酸(DNA和RNA)糖、蛋白质和脂类。后两者是这项拨款申请的主题。蛋白质分子是氨基酸的聚合物，折叠成定义的三维(3D)功能结构。例如，胶原蛋白在我们的大多数组织中提供支架；血红蛋白将氧气从肺部输送到活跃的器官；己糖激酶分解含葡萄糖的食物以帮助提供生物能量。许多蛋白质在水中折叠并发挥作用。从本质上讲，蛋白质中有两种氨基酸：一种是疏水性氨基酸，字面意思上是“憎水”；另一种是极性氨基酸，它可以溶于水。一种具有极性和疏水部分的水溶性蛋白质会折叠，将其大部分极性氨基酸放在其表面并与水接触，并将其大部分疏水氨基酸掩埋。然而，许多生物学活动发生在细胞膜之间或细胞膜内，这些不是简单的充满水的空间，需要一套不同的蛋白质。细胞周围的生物膜主要由脂分子组成。脂类也有两个截然不同的疏水区和极区。在膜中，许多脂质聚集在一起形成双层，其中一叶脂与另一叶脂相互作用，掩埋疏水部分，使极性部分暴露在水中；很像三明治，面包(在这个比喻中的极性部分)在外面，而馅料(疏水部分)在中间。这种组织在很大程度上制造了不渗透的屏障，这在生物学上是一个问题，需要其他分子，即跨膜蛋白质，来促进跨膜的运输和通信。自然界使用这些蛋白质来执行许多功能，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。跨膜蛋白质的整体化学与水溶性蛋白质不同；它们在外部和内部都是疏水的。这使得它们更难研究和理解。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，每条都折叠成一个螺旋。这些捆绑在一起形成一个圆柱体，上面有一个洞，有点像一堆马球薄荷糖。这种结构类似于称为通道的跨膜蛋白质。在这里，我们建议通过合理的蛋白质设计将水溶性的CC-Hex转变为跨膜蛋白。关键是我们了解CC-Hex的化学和结构，这将指导我们的设计。为什么要这样做？著名的物理学家理查德·费曼说过，他不能建造的东西，他就不明白。这是我们采用的原则：我们将研究天然的跨膜蛋白质，向它们学习，然后通过从CC-Hex设计简化的跨膜通道来测试我们的理解。这有可能行不通，但潜在的回报是高的：我们至少可以了解生物学的一些组成部分是如何组装的；也许我们可以利用这一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，一类被称为水通道蛋白的天然跨膜通道跨细胞膜运输和控制水的平衡。例如，在肾脏中，水通道蛋白从尿液中回收水分，将其浓缩，以帮助避免脱水。水通道蛋白是一种大而复杂的分子。如果我们能在CC-Hex这样的小蛋白中捕捉它们的性质，我们就有可能制造出在净水和海水淡化设备中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.