Directed Compartment Self-Assembly and Modulation of Encapsulin Quaternary Structure Öffentlichkeit

Abstract

Similar to membrane-based compartmentalization in eukaryotic cells, prokaryotes can establish physical boundaries within their cellular environment with the help of protein-based encapsulins. Individual encapsulin protomers can self-assemble to form nanocompartments; hollow spheres consisting of either 60 or 180 homomeric subunits. Structure analyses suggest that the E-loop, one of encapsulin’s three distinct structural features, is responsible for conformational changes which give rise to the two distinct quaternary structures of these nanocompartments. The structural effects of swapped E-loops of encapsulins from Thermotoga maritima (TmE; native 60-mer) and Myxococcus xanthus (MxE; native 180-mer) are explored to determine the effect of chimeragenesis on protein quaternary structure. The study of encapsulin protein chimeras revealed conserved N-terminal glycine and C-terminal proline residues that flank the E-loop, yet formation of these chimeric constructs suffered from compartment stability issues. Encapsulin shell structure is further explored through the steric-based obstruction of compartment self-assembly via lumen-oriented fusion of maltose binding protein (MBP). MBP-TmE fusion protein provides a novel method of in vitro shell assembly, termed “directed compartment self-assembly” (DCSA). MBP-TmE fusion protein studies revealed the discovery of arrested oligomeric states that precede the formation of a fully-assembled nanocompartment. These studies also elucidated a novel mechanism for in vitro self-assembly of encapsulin nanocompartments triggered through fusion protein cleavage under physiological conditions.

Table of Contents

1. List of Figures……..……………………………………………………………...…………….I 

2. Abbreviations …………………………………….……………………………………………II 

Chapter 1. Chimeragenesis to Modulate Encapsulin Quaternary Structure 

3. Background………………………………...…………………………………………………2-5 

4. Materials and Methods…….…………………………………………….……………....6-14 

5. Results and Discussion……...…………………………………………………….....…14-18 

Chapter 2. Directed Compartment Self-Assembly 

6. Background………………………………...………………………………………………20-22 

7. Materials and Methods…….…………………………………………………………….23-28 

8. Results and Discussion……...……..…..…………………………………………….…29-36 

9. Conclusions and Future Work ………………………………………………...……….36-38 

10. Primer Table………………………..………………………………..………………...…39-41 

11. References…………………………..………………………………..…………………...42-43 

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Stichwort	Protein Chimeragenesis Directed Compartment Self-Assembly Encapsulin Oligomer Icosahedron Quaternary Structure
Committee Chair / Thesis Advisor	Stefan Lutz, Emory University
Committee Members	Vincent Conticello, Emory University Yonggang Ke, Emory University

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