The Role of Water in "the Hydrophobic Effect:" Carbonic Anhydrase as a Model Protein for Physical-Organic Studies of Biomolecular Recognition

来自哈佛大学的George Whitesides博士将在分子水平上研究系统中疏水效应的细节，在这些系统中，熵占主导地位的观点（从疏水界面中有序水的释放）并不适用。许多蛋白质配体研究（理论和实验）表明，与疏水界面接触的水分子的焓和熵都很重要。这项工作考虑了蛋白质和配体之间相互作用的三个组成部分：蛋白质结合位点的表面，配体的结构，以及填充结合位点和包围配体的介质的组成。所选择的系统由芳基磺酰胺配体组成，它可以很容易地合成，与模型蛋白，人碳酸酐酶（HCA）相互作用。HCA的结构是已知的，研究人员有一个HCA与芳基磺胺配体配合物的晶体结构文库。有人会说，通常不被考虑的介质与蛋白质和配体一样重要，因为蛋白质和配体周围的水分子网络的结构将决定结合过程中发生的焓和熵的变化。本研究将结合热力学测量（等温滴定量热法）、结构分析（x射线晶体学和核磁共振）和分子模型来描述：(i) HCA、（ii）芳基磺酰胺配体和（iii）介质（我们将改变其组成和复杂性）之间的相互作用。这项工作解决了生物化学中被接受的两个“事实”：(i)疏水效应是一个熵主导的过程，在这个过程中有序的水从疏水界面中被排出；（ii）配体（或底物）与蛋白质（或酶）的结合是由两个分子的直接相互作用产生的，这是一个“锁与钥匙”的概念。这两个“真理”都很容易想象，因此很容易在入门课程中解释。这项工作质疑了这些“真相”，希望它能引起其他人质疑他们不仅研究疏水效应，而且研究分子识别的方法。一个成功的配体（在许多情况下是一个成功的“药物”）与它的靶标紧密结合；然而，我们无法以合理的方式预测紧密结合配体的结构。基础研究，如研究人员对蛋白质、配体和介质在蛋白质-配体结合中的作用的理解，为改进我们在合理配体设计中使用的过程提供了必要的信息。采用蛋白质活性位点水分子网络结构的模型将大大提高设计更紧密结合配体的能力。该项目积极地涉及本科生作为URI/REU学生，特别是从事各种物理测量的工作。他们在蛋白质表达和纯化方面也是同事：这个项目为学习蛋白质化学方面的高级技术技能提供了广泛的机会。怀特塞兹博士和他的同事们还在本科院校讲授这项工作，为生物物理学的当前概念提供了一个很好的介绍。这项研究的成果对制药行业很有兴趣，在制药行业，用于开发药物的过程已经变得非常昂贵，并且在蛋白质配体结合的计算和模拟以及“合理的先导开发”方面有了新的和积极的兴趣。

In this award from the Chemistry of Life Processes in the Chemistry Division, Dr. George Whitesides, from Harvard University, will study the molecular level-details of the hydrophobic effect(s) in systems where the entropy-dominated view (from the release of ordered water from a hydrophobic interface) does not apply. A number of protein-ligand studies (theoretical and experimental) suggest that both enthalpy and entropy of the water molecules in contact with a hydrophobic interface are important. This work considers each of the three components involved in the interaction between a protein and a ligand: the surface of the protein binding site, the structure of the ligand, and the composition of the medium that fills the binding site and surrounds the ligand. The system of choice consists of arylsulfonamide ligands, which can be easily synthesized, interacting with a model protein, human carbonic anhydrase (HCA). The structure of HCA is known, and the researchers have a library of crystal structures of complexes of HCA with arylsulfonamide ligands. The argument will be made that the medium, which is often not considered, is as important as the protein and the ligand, because the structure of the network of water molecules around both the protein and the ligand will determine the enthalpic and entropic changes that occur during binding. This study will combine thermodynamic measurements (isothermal titration calorimetry), structural analysis (X-ray crystallography and NMR) and molecular modeling to characterize the interactions between: (i) HCA, (ii) an arylsulfonamide ligand, and (iii) the medium (whose composition and complexity we will change). The work addresses two "truths" that are accepted in biochemistry: (i) the hydrophobic effect is an entropy-dominated process in which ordered waters are expelled from a hydrophobic interface and (ii) the binding of a ligand (or substrate) to a protein (or enzyme) results from the direct interaction of the two molecules, the notion of a "lock-and-key" fit. Both of these "truths" are easy to visualize and thus easy to explain in introductory courses. The work questions these "truths", with the hope it will cause others to question methods in which they approach not only the hydrophobic effect, but also molecular recognition. A successful ligand (and in many cases a successful 'drug') binds tightly to its target; however, we are unable to predict the structure of a tight-binding ligand in a rational manner. Fundamental studies, such as the researchers work on understanding the role of the protein, the ligand, and the medium in protein-ligand binding, provide the information necessary to improve the processes we use in rational ligand design. A model that takes the structure of the network of water molecules in the active site of the protein will, greatly improve the ability to design a tighter binding ligand.The project actively involves undergraduates as URI/REU students, especially working in a variety of physical measurements. They have also been coworkers in protein expression and purification: this project provides wide opportunity to learn high-level technical skills in protein chemistry. Dr. Whitesides and his co-workers also lecture on this work at undergraduate colleges, where it provides a good introduction to current concepts in biophysics. The output of this research is of interest in the pharmaceutical industry, where the processes used to develop drugs have become prohibitively expensive, and where there is renewed and active interest in computation and simulation of protein ligands binding and of "rational lead development."