Structure and Dynamics of an Evolved 3C Protease

描述(申请人提供)：运动和动力学对蛋白质功能的影响最近成为一个重要的研究领域。核磁共振光谱学的最新进展使分析和量化单个蛋白质残基的运动成为可能。脊髓灰质炎病毒3C蛋白水解酶是丝氨酸蛋白水解酶和微囊状病毒3C水解酶的模型，由于其相对较小的尺寸，为利用核磁共振波谱和mRNA展示研究功能和动力学提供了独特的机会。具体目标一：表征脊髓灰质炎病毒3C蛋白水解酶的动态和基本结构特性，该水解酶是丝氨酸蛋白酶的模型。利用2H和15N弛豫实验研究了野生型3C的动力学性质。这一目标将提供有关该酶的功能动力学和分子内通讯的信息。特定目标II：利用定向进化方法进化具有增强活性的脊髓灰质炎病毒3C蛋白。定向进化和mRNA展示将被用于进化和筛选比野生型蛋白具有更高活性效率的3C蛋白酶。将对表现出增强活性的进化的蛋白酶进行筛选，以识别那些突变不会直观地影响功能的酶。具体目的III：绘制3C酶的进化位点和活性位点之间的功能连接性图谱。这一目标的目的将是将进化的蛋白酶与野生型蛋白质进行比较。野生型的动态和基本结构变化将使用在AIM I中进行的相同的2H和15N弛豫实验来监测。对动力学中的任何变化的检查都可能导致发现对蛋白质功能重要的神秘的能量途径。这项研究的直接目标是监测具有增强活性的进化的蛋白水解酶的动态和结构变化。进一步的目标包括使用定向进化来选择特异性的变化，以及进化出更稳定的蛋白酶。通过观察这些稳定性和特异性的变化，可能会阐明有助于根除某些疾病的新的药物靶点。相关性：3C蛋白水解酶是微小核糖核酸病毒生命周期中的一个基本成分，这使得它成为抗病毒治疗的一个有吸引力的靶点。它也是丝氨酸蛋白酶的模型，丝氨酸蛋白酶是一种蛋白质，在整个身体的许多过程中发挥着至关重要的作用。这项研究中的比较可能会给蛋白质设计带来新的洞察力，也可能导致药物和抗病毒疗法的新靶点，而这些靶点可能是传统方法所没有发现的。

DESCRIPTION (provided by applicant): The effect of motion and dynamics on protein function has recently emerged as an important area of research. Recent advances in NMR spectroscopy have allowed the analysis and quantification of motions of individual protein residues. The poliovirus 3C protease, a model for both serine proteases and the piconovirus 3C protease, presents a unique opportunity of study of function and dynamics using NMR spectroscopy and mRNA display because of its relatively small size. Specific Aim I: Characterize the dynamic and basic structural properties of the poliovirus 3C protease, a model for serine proteases, in solution. 2H and 15N relaxation experiments will be utilized to study the dynamic properties of wild-type 3C. This aim will provide information about the functional dynamics and intramolecular communication of the protease. Specific Aim II: Evolve poliovirus 3C protease proteins with enhanced activity using directed evolution. Directed evolution and mRNA display will be used to evolve and select for 3C proteases with higher efficiencies of activity than the wild-type protein. Evolved proteases that exhibit enhanced activity will be screened to identify those with mutations that would not intuitively affect function. Specific Aim III: Map functional connectivities between the evolved sites of 3C protease and the active site. The goal of this aim will be to compare the evolved proteases with the wild-type protein. Dynamic and basic structural changes from the wild-type will be monitored using the identical 2H and 15N relaxation experiments performed in Aim I. Examination of any changes in dynamics could lead to the discovery of cryptic energetic pathways that are important for protein function. The immediate goal of this study is to monitor the dynamic and structural changes in evolved proteases with enhanced activity. Further goals include using directed evolution to select for changes in specificity as well as evolving more stable proteases. Observations from these changes in stability and specificity may elucidate novel drug targets that assist in the eradication of certain diseases. Relevance: The 3C protease is an essential element in the life cycle of picornaviruses which make it an attractive target for antiviral therapy. It is also a model for serine proteases which are proteins that play vital roles in many processes throughout the body. Comparisons made in this study could give new insight into protein design and could also lead to new targets for both pharmaceutical and antiviral therapies that may not have been discovered through conventional means.