發展以結構生物資訊為基礎之立體域交換蛋白質工程平台

Abstract

蛋白質立體域交換現象(3D domain swapping)是蛋白質形成四級結構的一種機制。其定義 為二或多個單體(monomer)蛋白質彼此交換相同的域(domain)以形成結構交錯的聚集體 (oligomer)。研究此現象有助人們了解蛋白質的功能調控、分子演化與結構動力學特性， 且有利開發生物性聚合物及尋找蛋白質堆積性疾病如阿茲海默症與狂牛症的療法。儘管 立體域交換現象漸受關注，目前仍無系統化的工具得以大量辨識出此類蛋白。連接具此 現象的蛋白質的兩個域的片段稱為hinge loop。研究 hinge loop 有助深入了解此現象， 但可準確算出其位置與範圍的方法極少。在此計畫中，我們決定開發第一個立體域交換 現象結構搜尋比對系統，名為DSSARST，並以之為基礎建立域交換現象資料庫。其中 資訊將使我們得以開發預測工具來指導蛋白質工程，轉變單體為聚集體或反之，並提升 其活性。最後，應用所開發的預測方法，實際以生物學技術來改進某些可用於生產生殖 能源的酵素之活性。本計畫申請人羅惟正與蘇士哲博士的主要研究領域為結構生物學與 蛋白質工程。基於過去愉快的合作默契與互補的專業能力，他們有信心能完成此計畫書 所提的諸多工作。而過程中所開發的計算生物學方法、軟體與功能新穎的蛋白質，將大 有力推動域交換現象之研究及其於生物工程上的應用。

Three-dimensional domain swapping (DS), a structural phenomenon first clearly described in the mid 1990s, is a mechanism for protein oligomerization defined as two or more proteins exchanging part of their identical domain to form intertwined oligomers. Studying DS can help reveal the functional regulation, molecular evolution and structural dynamics of proteins, and help find applications in creating artificial biopolymers and treating deposition diseases like the Alzheimer’s disease and bovine spongiform encephalopathy. Despite the increasing interest in DS, related bioinformatics methods are rarely available and there is still a lack of a comprehensive database for studying DS. The domains of a DS protein are connected by a hinge loop. Hinge loops have been proposed to be the causes and/or the results of DS; however, precise methods for their identification are not readily available. We have previously developed a rapid protein structural similarity search method called SARST and an accurate method ADiDoS for the detection of DS relationship between proteins. In this proposal, we have determined to improve and combine them into the first database searching system for DS-related proteins. This searching system, named DSSARST (3D Domain Swapping Search Aided by Ramachandran Sequential Transformation), will enable large-scale studies on this interesting phenomenon and facilitate its application in biotechnology. By utilizing DSSARST, we will then establish the first DS protein database, which is going to provide plenty information for researchers and us to elucidate the mechanisms of DS and the properties of the hinge loop. Results of these studies will further enabled us to develop prediction systems determining whether and how a protein can be artificially engineered to become a domain-swapped oligomer or monomer and thus acquires improved functionality. We will finally apply our prediction systems and use wet-lab procedures, including mutagenesis, protein expression and purification, and structural determination with nuclear magnetic resonance (NMR) spectroscopy, to engineer several important enzymes used to produce biofuels. The research interests of the applicants, Dr. Wei-Cheng Lo and Dr. Shih-Che Sue, mainly focus on structural biology and protein engineering. Lo had received solid biochemistry wet-lab trainings in college and greatly expanded his capability of doing computational biology researches in his doctoral days. Dr. Sue is an experimental structural biologist with many experiences in cooperating with computational biologists. They have collaborated for three years in an Interdiscipline Project funded by the National Science Council. Because of their pleasant experiences in previous collaborations and their complementary professional backgrounds, they are supposed fully capable of accomplishing the proposed research project, which shall be greatly helpful for moving DS related researches and bioengineering fields forward.