Dynamic hydrogels: Translating a protein conformational change into macroscopic motion
Dynamic hydrogels: Translating a protein conformational change into macroscopic motion
复制标题
DOI：
10.1002/anie.200604808
复制
发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Mrksich, Milan
中科院分区：
文献类型：
--
作者：
Murphy, William L.;Dillmore, W. Shannon;Mrksich, Milan
经历形状变化的材料的发展占据了材料科学的中心主题，并在许多应用中被证明是重要的。有几种水凝胶是交联水溶性聚合物，可以根据温度、ph值或离子强度改变其性质（如体积、交联密度）。[1-5]这些动态水凝胶也可以用生化部分修饰，从而得到响应蛋白质和配体而改变其性质的材料。[3,6]例如，由于抗原-抗体[2]和凝集素-碳水化合物[7,8]相互作用而发生体积变化的水凝胶已被用作生物传感器。动态水凝胶材料的基本工作原理涉及其物理或化学交联密度的变化，以响应环境线索。这些方法的一个未开发的替代方案依赖于使用一种天然蛋白质，这种蛋白质会发生构象变化，作为改变材料特性的机制。蛋白质运动在生物系统中的功能重要性，以及可以利用的广泛的蛋白质运动，为制备动态材料提供了灵活的方法。我们在这里描述了一个基于蛋白质的动态材料的例子，其功能性质来源于蛋白质钙调素（CaM）的构象性质。钙调素是一种16.5 kda的蛋白，具有两种不同的构象状态（图1）。[9-13]在钙离子存在下，CaM呈延伸的哑铃状构象（此处称为“延伸的CaM”）这种钙结合的CaM在结合配体（包括某些抗精神病药物（如三氟拉嗪（TFP））、[10,13,16]肽、[9]和各种蛋白质[12]）后，经历从扩展哑铃到崩溃构象（这里称为“崩溃的CaM”）[15]的转变。最近，Daunert和同事描述了一类水凝胶，它将CaM和一个小分子配体作为网络中的垂坠部分CaM单元和配体的结合增加了交联密度，减少了网络的膨胀。这种方法类似于基于抗原-抗体[2]和凝集素-碳水化合物[7,8]相互作用的动态水凝胶的开发，但与我们的方法不同的是，凝胶的动态响应主要不是由于CaM的构象性质。我们制备了一个工程版本的CaM，其中在哑铃状蛋白（CaMY34C, Y110C）的末端包含半胱氨酸残基代替酪氨酸残基。在伸展构象中，两个半胱氨酸残基之间的距离约为50，而在折叠构象中，这一距离约为15我们通过使用四臂聚乙二醇（PEG）分子将CaM构建块整合到聚乙二醇基水凝胶中，每个末端都有一个丙烯酸酯基团。丙烯酸酯基团选择性地与工程CaM上的巯基反应，从而使CaM蛋白交联成水溶性偶联物。测量游离巯基数量的Ellman测试在5分钟后进行，结果表明PEG四丙烯酸酯与工程CaM的反应完成（图2a）。此外，MALDI-TOF质谱分析证实了游离CaM蛋白转化为交联产物（图2b）。例如，MALDI光谱在% 27 kDa处显示了一个漫射峰，这代表了单个PEG四丙烯酸酯的共轭物。
The development of materials that undergo shape changes occupies a central theme in materials science and has proven important in several applications. Several classes of hydrogels, which are cross-linked water-soluble polymers, can change their properties (such as, volume, cross-link density) in response to temperature, pHvalue, or ionic strength.[1–5] These dynamic hydrogels can also be modified with biochemical moieties to give materials that change their properties in response to proteins and ligands.[3, 6] For example, hydrogels that undergo volume changes because of antigen–antibody [2] and lectin–carbohydrate [7, 8] interactions have been used as biosensors. The underlying principle of operation of dynamic hydrogel materials relates to a change in their physical or chemical cross-linking density in response to environmental cues. An unexplored alternative to these approaches relies on the use of a natural protein that undergoes a conformational change as a mechanism to alter the characteristics of a material. The functional importance of protein motions in biological systems, together with the wide range of protein motions that can be harnessed, offers a flexible approach to the preparation of dynamic materials. We describe herein an example of a protein-based dynamic material, the functional nature of which is derived from the conformational properties of the protein calmodulin (CaM). Calmodulin is a 16.5-kDa protein with two distinct conformational states (Figure1).[9–13] In the presence of calcium ions, CaM has an extended, dumbbell-shaped conformation (herein termed “extended CaM”).[14] This calciumbound CaM undergoes a transition from an extended dumbbell to a collapsed conformation (herein termed “collapsed CaM”)[15] upon the binding of ligands, which include certain antipsychotic drugs (such as, trifluoperazine (TFP)),[10, 13, 16] peptides,[9] and a variety of proteins [12]. Recently, Daunert and co-workers described a class of hydrogels that incorporate CaM and a small-molecule ligand as pendant moieties within the network.[17] The binding of the CaM units and ligands resulted in an increased cross-linking density and a decreased swelling of the network. This approach is analogous to the development of dynamic hydrogels based on antigen–antibody [2] and lectin–carbohydrate [7, 8] interactions, but differs from our approach in that the dynamic response of the gel is not due primarily to the conformational properties of CaM. We prepared an engineered version of CaM in which cysteine residues are included in place of tyrosine residues at the ends of the dumbbell-shaped protein (CaMY34C, Y110C). The distance that separates the two cysteine residues is approximately 50 in the extended conformation,[18] but is decreased to approximately 15 in the collapsed conformation.[16] We incorporated the CaM building blocks into a poly (ethylene glycol)-based hydrogel by using a four-armed poly (ethylene glycol)(PEG) molecule terminated at each end with an acrylate group. The acrylate groups react selectively with the sulfhydryl groups on the engineered CaM and therefore serve to cross-link the CaM proteins into watersoluble conjugates. The Ellman test, which measures the amount of free sulfhydryl groups, was performed after a period of 5 minutes and showed that the reaction of the PEG tetraacrylate with the engineered CaM was complete (Figure 2a). Furthermore, MALDI-TOF mass spectrometry confirmed the conversion of the free CaM protein into crosslinked products (Figure 2b). The MALDI spectra, for example, showed a diffuse peak at% 27 kDa, which represents a conjugate of a single PEG tetraacrylate …