Structure and Dynamics of an Evolved 3C Protease

进化的 3C 蛋白酶的结构和动力学

• 批准号: 7663858
• 负责人: Chad Petit
• 金额: $ 4.96万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7663858
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Affect; Antiviral Agents; Antiviral Therapy; Area; Biological Models; Catalysis; Communication; Cysteine Protease; Disease; Drops; Drug Delivery Systems; Encephalomyocarditis virus; Endopeptidases; Enzymes; Exhibits; Family; Family Picornaviridae; Foot-and-Mouth Disease Virus; Future; Genetic Identity; Genotype; Goals; HIV; Hepatitis A Virus; Human poliovirus; In Vitro; Indium; Individual; Knowledge; Lead; Life Cycle Stages; Link; Maps; Messenger RNA; Methods; Modeling; Monitor; Motion; Mutagenesis; Mutation; NMR Spectroscopy; Pathway interactions; Peptide Hydrolases; Pharmacologic Substance; Phenotype; Play; Pliability; Polioviruses; Polymerase Chain Reaction; Process; Property; Protease Inhibitor; Protein Region; Proteins; Purpose; Relaxation; Research; Rhinovirus; Role; Serine; Serine Protease; Side; Site; Solutions; Specificity; Structure; Techniques; Time; Vertebral column; Viral; Virus; Virus Diseases; Work; chemotherapeutic agent; design; directed evolution; insight; member; mutant; new technology; novel; pressure; prevent; protein function; research study; size

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.

描述（由申请人提供）：运动和动力学对蛋白质功能的影响最近已成为一个重要的研究领域。核磁共振波谱学的最新进展可以对单个蛋白质残基的运动进行分析和定量。脊髓灰质炎病毒 3C 蛋白酶是丝氨酸蛋白酶和小核糖核酸病毒 3C 蛋白酶的模型，由于其相对较小的尺寸，为使用 NMR 波谱和 mRNA 显示研究功能和动力学提供了独特的机会。具体目标 I：表征溶液中脊髓灰质炎病毒 3C 蛋白酶（丝氨酸蛋白酶的模型）的动态和基本结构特性。 2H和15N弛豫实验将用于研究野生型3C的动态特性。该目标将提供有关蛋白酶的功能动力学和分子内通讯的信息。具体目标 II：利用定向进化进化出具有增强活性的脊髓灰质炎病毒 3C 蛋白酶蛋白。定向进化和mRNA展示将用于进化和选择比野生型蛋白质具有更高活性效率的3C蛋白酶。将筛选表现出增强活性的进化蛋白酶，以识别那些不会直观地影响功能的突变的蛋白酶。具体目标 III：绘制 3C 蛋白酶进化位点与活性位点之间的功能连接图。该目标的目的是将进化的蛋白酶与野生型蛋白质进行比较。将使用 Aim I 中进行的相同 2H 和 15N 弛豫实验来监测野生型的动态和基本结构变化。对动力学的任何变化的检查可能导致发现对蛋白质功能很重要的神秘能量途径。这项研究的直接目标是监测活性增强的进化蛋白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。对这些稳定性和特异性变化的观察可以阐明有助于根除某些疾病的新药物靶点。相关性：3C 蛋白酶是小核糖核酸病毒生命周期中的重要元素，这使其成为抗病毒治疗的有吸引力的靶标。它也是丝氨酸蛋白酶的模型，丝氨酸蛋白酶是在全身许多过程中发挥重要作用的蛋白质。这项研究中进行的比较可以为蛋白质设计提供新的见解，也可以为药物和抗病毒疗法带来通过传统手段可能无法发现的新靶点。