Zhang, Deqiang (2003) Structure-based design of mutant proteins: I. Molecular docking studies of amino acid binding to wild-type aminoacyl-tRNA synthetases. II. Structure-based design of mutant aminoacyl-tRNA synthetases for non-natural amino acid incorporation. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-12182002-190040
Protein biosynthesis has precisely controlled accuracy, and aminoacyl-tRNA synthetases (AARSs) play an important role in charging amino acids to their cognate tRNAs with high fidelity. In some cases the misactivation of non-natural amino acids by the wild-type or mutant AARS can be utilized to incorporate these non-natural amino acids into proteins in vivo. Such technique has tremendous potentials in protein engineering and other applications. Therefore, it is essential to understand the amino acid recognition mechanism displayed by AARSs. In this thesis, computational studies of the selection of natural and non-natural amino acids by AARSs at the binding stage have been conducted for methionyl-tRNA synthetase (Chapter 2), histidyl-tRNA synthetases (Chapter 3), and isoleucyl-tRNA synthetase (Chapter 4). In these chapters, molecular docking and ligand perturbation are used to elucidate the binding discrimination showed by these AARSs. Because many non-natural amino acids carrying interesting physical and chemical properties on their side chains cannot be incorporated by using the wild-type AARSs, it is necessary to manipulate the activity of AARSs by making mutations in the binding site of amino acids. To this end, we have developed a Clash Opportunity Progressive (COP) protein design tool to redesign the binding site of AARSs. Chapter 5 describes the main steps in COP. Chapters 6 to 8 present the application of COP to different AARSs. In Chapter 6, COP has been applied to design mutant tyrosyl-tRNA synthetase (TyrRS) for recognizing Ome-Tyr, Naph-Ala, and p-keto-Tyr. In Chapter 7, COP has been used to design mutant phenylalanyl-tRNA synthetase for p-keto-Phe. In Chapter 8, tryptophanyl-tRNA synthetase is used as a template to design mutant AARS to recognize NBD-Ala, bpy-Ala, and DAN-Ala. The appendices are some publications and manuscripts on various other projects. Appendix I is a molecular dynamics study of laboratory-evolved pNBE enzymes with different thermostability. The findings presented here will help us to better understand the determinants in protein stability evolution. Appendix II contains experimental work I have done in the Chan group. Unfolding experiments revealed the existence of intermediates in the equilibration unfolding of RdPf. In Appendix III, femtosecond time-resolved spectroscopy was used to study the fluorescence resonance energy transfer and tryptophan solvation dynamics in RdPf.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||COP; ligand perturbation; molecular docking; non-natural/unnatual amino acid incorporation; protein design|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||17 December 2002|
|Author Email:||zhangdq (AT) wag.caltech.edu|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||19 Dec 2002|
|Last Modified:||26 Dec 2012 03:14|
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