핵자기공명분광법을 이용한 EDB와 EDB 앱타이드 복합체의 backbone assignment

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부(바이오모듈레이션전공), 2014. 2. 윤철희.The extra domain B (EDB) of fibronectin, a naturally occurring marker of tissue remodeling and angiogenesis, is expressed in the majority of aggressive solid human tumors, whereas it is not detectable in normal vessels and tissues. Aptides based on the tryptophan zipper scaffold with variable target-binding arms were shown to recognize diverse target proteins with high affinity and specificity. I employed NMR spectroscopy in order to characterize the binding mode of EDB and its specific aptide. Performed three-dimensional triple resonance NMR experiments to assign the backbone resonances of free EDB and EDB:aptide complex using double labeled (13C/15N) and triple labeled (2H/13C/15N) samples. 3D CBCACONH, HNCACB, and HBHA(CO)NH were recorded and analyzed, yielding a total of 97% of the 1Hα, 13Cα, and 13Cβ chemical shift assignment. After that I calculated the Chemical Shift Index (CSI) using the backbone chemical shifts. The results indicated that six β-strand secondary structures were found between residues 5-15, 20-28, 34-42, 51-55, 62-64, and 7280 and also a α-helical turn between residues 5659. Comparison of the CSI between free EDB and the EDB:aptide complex revealed a dramatic change in the secondary structures upon the complex formation. Based on the backbone chemical shift assignment, side chain assignment and distance restraint measurement are underway to determine the three-dimensional structure of the complex.ABSTRACT CONTENTS LIST OF FIGURES LIST OF TABLES ABBREVIATIONS I. INTRODUCTION 1.1. Extra domain B 1.2. Aptide 1.3. Principles of backbone assignment by NMR spectroscopy 1.4. Chemical Shift Index II. MATERIALS AND METHODS 2.1. Protein expression and purification 2.2. NMR experiments III. RESULTS AND DISCUSSION 3.1. Sample preparation of EDB and aptide 3.2. NMR assignment IV. CONCLUSION V. REFERENCES ABSTRACT IN KOREANMaste