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Control of Enzyme Activity Using Chemical Rescue of Protein Structure

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Control of Enzyme Activity Using Chemical Rescue of Protein Structure Control of Enzyme Activity using Chemical Rescue of Protein Structure by Yang Du A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering Baltimore, Maryland May 2016 ii Abstract A cell can modulate the level of protein activity by the regulation of the amount of protein. On the other hand, strategies for controlling of cellular protein activity may lie in regulating the protein’s specific activity directly. Based on this idea, we demonstrated a method for designing an effector site directly into the catalytic domain of an enzyme and using a chemical compound to regulate the protein’s activity. This approach is different from the traditional chemical rescue of enzymes because it relies on disruption and restoration of protein structure instead of disrupting the active site and having the chemical compound restore the chemical functionality to the active site as a means to achieve modulated function. In detail, we demonstrate the activity of TEM-1 can be disrupted by removing buried tryptophan or leucine side chain that serve as a buttress supporting the protein structure. Three cases of site-direct mutations (W227G & W286G, L247G & W286G, and L282G & W286G) to TEM-1 β-lactamase enzyme (BLA) were made. In all cases, we observe disruption of enzyme function. To test the theory of chemical rescue of protein structure, we then assay β-lactamase activity in the presence of specific chemical compounds designed to bind in place of the iii side chains of the mutated amino acids. We observed an 8- fold restoration of W227G/W286G enzyme function in the presence of 2- (1-naphthylmethyl)-isoquinoline (NMIQ) and 3 -methyl-2- (1-naphthylmethyl)-1,3-benzothiazol-3- ium (MNMBT). The other two chemicals, however, don’t have a noticeable impact on enzyme activity. The results provide a new example of now it is possible to use chemical rescue of protein structure to create regulatable enzymes. Chemical rescue of protein structure can, therefore, be a potentially general useful method for regulating enzyme activity. Advisor: Professor Marc Ostermeier Readers: Professor Marc Ostermeier Professor Jeff Gray iv Acknowledgement Here I want to take this chance to express my gratitude to all the people who helped me for the past two years. I would like to thank especially to Dr. Marc Ostermeier, who gave me the opportunity to get access to biomolecular technologies. Thanks to his priceless guidance, my research reached to a higher level. I would also like to thank all the lab members. Special thanks go to Nathan Nicholes and Lucas Ribeiro for being great colleagues and friends in the lab. Without their help, this research wouldn’t be done successfully. Their kindness supported me to go through these days. I would like to say thanks to my classmates, Xiaotong, Tianyu, and Xinpei. Without their encouragement, I couldn’t adhere to my academic life and go so far. At last, I appreciate all the supports from my parents and cousin. They back up my life and stay with me no matter what I am facing. Their loves make up the best part of my life v vi Table of Contents Abstract .............................................................................................................................. iii Acknowledgement .............................................................................................................. v Table of Contents .............................................................................................................. vii List of Figures .................................................................................................................... ix Chapter 1 Introduction and Background ............................................................................. 2 1.1 Switchable Enzyme ............................................................................................... 2 1.2 Design of Switchable Enzyme .............................................................................. 3 1.2.1 Protein Fragment Complementation Assays ............................................................... 4 1.2.2 Proteins fused with overlapping sequences ................................................................ 5 1.2.3 Circular Permutation ................................................................................................... 6 1.3 Chemical Complementation of Reengineered Enzyme ........................................ 7 1.4 Chemical Rescue of Protein Structure .................................................................. 9 Chapter 2 Theory of Chemical Rescue of Protein Structure and Design of the Experiment ........................................................................................................................................... 12 2.1 Mechanisms of Inactivation ................................................................................ 12 2.2 Selection of Cavity-Forming Site and Complement Chemical Compound ........ 14 2.3 Design for mutated TEM-1 β-lactamase ............................................................. 16 2.4 Chemical Compound Selection........................................................................... 18 Chapter 3 Materials and method ....................................................................................... 22 vii 3.1 Creation of Variants ............................................................................................ 22 3.2 MIC Test for Wild Type and Variants ................................................................. 24 3.3 Chemical Compounds Preparation ..................................................................... 25 3.4 Chemical Rescue of Variants .............................................................................. 26 3.5 Additional Test .................................................................................................... 26 Chapter 4 Result and Discussion ...................................................................................... 30 4.1 Enzyme Inactivation ........................................................................................... 30 4.2 Enzyme Re-activation ......................................................................................... 32 4.3 Chemical Specificity ........................................................................................... 37 4.4 Preparation for Protein Purification .................................................................... 39 Chapter 5 Future work ...................................................................................................... 42 5.1 Purification of Protein ......................................................................................... 42 5.2 Quantifying Chemical Rescue of Protein Structure ............................................ 42 Chapter 6 Conclusion ........................................................................................................ 45 References ......................................................................................................................... 46 Appendix ........................................................................................................................... 49 Curriculum Vitae ............................................................................................................... 50 viii List of Figures Figure 2.1 Mechanisms of Inactivation ................................. 13 Figure 2.2 I ndole restores structure of enzyme ............................ 14 Figure 2.3 1XPB structure of TEM-1 beta-lactamase from E. coli. ............ 18 Figure 2.4 Computational models of all mutant–compound complexes ......... 19 Figure 4.1 The effect of the mutations on ampcillin resistance ............... 31 Figure 4.2.1 The effect of the chemical compounds and solution .............. 34 Figure 4.2.2 Ability of chemical compounds to rescue antibiotic resistance ..... 35 Figure 4.3 NMIQ or MNMBT rescue L247G/W286G and L282G/W286G ...... 38 Figure 4.5 TEM-1 β-lactamase maltose binding protein fusion ............... 40 ix Chapter 1: Introduction and Background 1 Chapter 1 Introduction and Background Proteins are complex molecules that play essential roles in cells. Mostly, they work in cells, and some are involved in structural support while others are involved in a cellular movement. Each protein is constructed of a different sequence from a set of 20 amino acids, which determines its function. Having a better understanding of proteins’ function is crucial on a cellular level for application in protein engineering. One area of protein engineering focuses on the manipulation of proteins to control cell function, signal transduction, growth, and survival through the use of protein switches. This type of protein engineering may include protein manipulation, production, and degradation through small-molecule-dependent switches that regulate transcription [Buskirk et al. 2005] or engineering the artificial gene regulatory networks [Kaern et al. 2003] . Another manipulation strategy refers to the construction of switchable enzymes [Kanwar et al. 2013] , which shows great promise for direct control of protein’s activity. 1.1 Switchable Enzyme Typically, a switchable enzyme can be defined as one in which an input signal through binding or modifications at one site alters the enzyme function at another site. This type of switchable enzymes have spatially different regulatory and active sites. Since changes at one site of an enzyme may affect
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