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Protein Engineering of Structurally Homologous Proteins

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Protein Engineering of Structurally Homologous Proteins The Pennsylvania State University The Graduate School PROTEIN ENGINEERING OF STRUCTURALLY HOMOLOGOUS PROTEINS A Thesis in Integrative Biosciences by Hui Li © 2005 Hui Li Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2005 The thesis of Hui Li was reviewed and approved* by the following: Stephen J. Benkovic Evan Pugh Professor and Eberly Chair in Chemistry Thesis Advisor Chair of Committee Ming Tien Professor of Biochemistry Squire Booker Associate Professor of Biochemistry and Molecular Biology Costas D. Maranas Professor of Chemical Engineering Richard J. Frisque Professor of Molecular Virology Co-Director, Graduate Education Integrative Biosciences Graduate Program The Huck Institutes of the Life Sciences *Signatures are on file in the Graduate School iii ABSTRACT One of the ultimate goals of protein engineering is the de novo design of novel proteins with desired activities and properties. However, our current knowledge of protein structures and functions are far from complete to achieve this goal. On the other hand, nature has successfully evolved an enormous number of proteins with novel functions for their hosts to fit the ever changing environments, and naturally occurring proteins present the most diverse and complicated information about protein structure- function relationships. Studying the evolutionary-related proteins with structural and functional homology will provide not only the detailed information about protein structure-function relationships, but also the insights to the strategies that nature had adopted for protein evolution. Here, we studied two pairs of enzymes with significant homology on their structures, reaction mechanisms, and the active site architectures. In our attempt to interconvert the enzymatic activities between two members in the same pair, rational methods, such as site-directed mutagenesis and rational domain swapping, were applied first on the basis of our current understanding of each protein. Further, the additional sequence spaces were explored using combinatorial methods, such as ITCHY, random mutagenesis, and DNA shuffling, to identify their potential roles in terms of protein structures and functions. The first pair of enzymes we chose are Escherichia coli purT-encoded glycinamide ribonucleotide (GAR) transformylase (PurT) and Escherichia coli N5- carboxylaminoimidazole ribonucleotide (N5-CAIR) synthetase (PurK). While both iv enzymes are involved in the de novo purine biosynthesis, PurT catalyzes the third reaction of the purine biosynthetic pathway, the conversion of GAR, ATP and formate to formyl GAR, ADP and inorganic phosphate (Pi); and PurK catalyzes the sixth reaction of the purine biosynthetic pathway, the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP and bicarbonate to N5-CAIR, ADP and Pi. The effort to interconvert the enzymatic activities between PurT and PurK suggested that these two enzymes might evolve through domain swapping. Several crucial structural elements for catalysis were also identified in each protein, which provides value information for protein structure- function relationships. The second pair of enzymes are Escherichia coli N-acetylneuraminate lyase (NAL) and Escherichia coli dihydrodipicolinate synthase (DHDPS), two (β/α)8 barrel proteins. NAL catalyzes the degradation of N-acetylneuminate (NANA) to N- acetylmannosamine (ManNAc) and pyruvate. DHDPS catalyzes the branch-point reaction of the lysine biosynthetic pathway in plants and microbes: the condensation of L- aspartate-β-semialdehyde and pyruvate to dihydrodipicolinate (DHDP). Both enzymes were observed to be able to catalyze each other’s reaction, and this functional promiscuity between NAL and DHDPS is considered as a strong statement that they are evolutionary-related. A possible evolutionary scheme from NAL to DHDPS through divergent path was further approved by the attempt to interconvert the enzymatic activities between NAL and DHDPS. A conserved Arg residue in DHDPS was identified to be crucial for the DHDPS activity. Several DHDPS mutants with an enhanced NAL activity were also identified using combinatorial methods. v TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................vii LIST OF TABLES.......................................................................................................x LIST OF ABBREVIATIONS......................................................................................xi ACKNOWLEDGEMENTS.........................................................................................xiv Chapter 1 Introduction ................................................................................................1 1.1 Protein Engineering ........................................................................................1 1.2 Protein Evolution............................................................................................4 1.3 Methodologies of Protein Engineering...........................................................7 1.3.1 Rational Protein Design........................................................................7 1.3.2 Combinatorial Approaches...................................................................13 1.3.2.1 Generation of Molecular Diversity for Directed Evolution .......14 1.3.2.2 Screening and Selection .............................................................25 Chapter 2 Rational Domain Swapping between purT-encoded GAR transformylase (PurT) and N5-CAIR synthetase (PurK) ......................................30 2.1 Introduction.....................................................................................................30 2.1.1 GAR transformylase (PurT) and N5-CAIR synthetase (PurK).............31 2.1.2 Protein evolution by domain swapping ................................................43 2.2 Experimental:..................................................................................................49 2.2.1 Materials:.............................................................................................49 2.2.2 Bacterial Strains: ..................................................................................50 2.2.3 Methods:...............................................................................................50 2.3 Results and Discussion ...................................................................................57 2.4 Conclusion ......................................................................................................73 Chapter 3 Identification of functional subdomains in purT-encoded GAR transformylase (PurT) and N5-CAIR synthetase (PurK) by combinatorial and rational methods....................................................................................................75 3.1 Introduction.....................................................................................................75 3.2 Experimental:..................................................................................................77 3.2.1 Materials:..............................................................................................77 3.2.2 Bacterial Strains: ..................................................................................78 3.2.3 Methods:...............................................................................................78 3.3 Results and Discussion ...................................................................................85 3.4 Conclusions: ...................................................................................................110 vi Chapter 4 Interconversion of enzymatic activities between N-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS), two (β/α)8 barrel proteins .................................................................................................................114 4.1 Introduction: ...................................................................................................114 4.1.1 The (β/α)8 barrel proteins......................................................................114 4.1.2 The evolution of the (β/α)8 barrel proteins ...........................................117 4.1.3 Protein engineering of the (β/α)8 barrel proteins..................................126 4.1.4 N-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS) ................................................................................................129 4.1.4.1 Introduction of NAL and DHDPS..............................................129 4.1.4.2 The active sites of NAL and DHHPS.........................................138 4.2 Experimental:..................................................................................................145 4.2.1 Materials:.............................................................................................145 4.2.2 Bacterial Strains: ..................................................................................146 4.2.3 Methods:...............................................................................................146 4.3 Results and Discussion ...................................................................................160 4.4 Conclusion ......................................................................................................190 Bibliography ................................................................................................................193 vii LIST OF FIGURES Figure 1: Schematic
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