Combinatorial approaches to protein folding and function

蛋白质折叠和功能的组合方法

• 批准号: 6608203
• 负责人: THOMAS J MAGLIERY
• 金额: $ 4.64万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6608203
• 关键词: X ray crystallography; binding sites; biotechnology; calorimetry; circular dichroism; conformation; flow cytometry; gene expression; green fluorescent proteins; metalloenzyme; postdoctoral investigator; protein binding; protein engineering; protein folding; protein structure function

DESCRIPTION: (provided by applicant) Folding and stability of proteins have emerged as key elements in the molecular understanding of diseases like cancer and Alzheimer?s, yet a complete understanding of these phenomena remains elusive. One highly productive approach has been the systematic redesign of proteins. A significant limitation to redesign is the characterization of the enormous number of possible variants that can be engineered. Methods for the analysis of large libraries of variants of well-studied proteins are highly desirable. An extremely well-studied model for protein folding and stability is the four-helix bundle protein Rop, which modulates ColE 1 plasmid copy number. This will serve as the basis for a screen for Rop function, wherein the production of a marker like green fluorescent protein from a ColE 1 plasmid reflects the integrity of a Rop protein variant. Such a screen will be used to identify active Rop variants from large libraries containing exhaustive mutations in loop or core residues. One interesting problem is the identification of parameters that control overall conformation. Recent experiments show that proteins can fold into highly disparate threedimensional structures despite high homology (for example, a variant of the alpha/beta B1 domain folds into the coiled-coil structure of Rop). Rop-like structures have been stabilized by metal ions through the engineering of novel metal-binding sites. We will examine the limits of conformational switching by using metal binding to stabilize a Rop-like structure from a B1 variant. We are also interested in isolating B1 variants that recapitulate Rop function. A screen for Rop function will allow us to radically expand the scope of protein redesign and refine our understanding of the parameters that control protein folding and stability.

描述：（由申请人提供）蛋白质具有折叠性和稳定性