The Pennsylvania State University The Graduate School COMPUTATIONAL REDESIGN OF CHANNEL PROTEINS, ENZYMES, AND ANTIBODIES A Dissertation in Chemical Engineering by Ratul Chowdhury © 2020 Ratul Chowdhury Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2020 The dissertation of Ratul Chowdhury was reviewed and approved* by the following: Costas D. Maranas Donald B. Broughton Professor of Chemical Engineering Dissertation Advisor Chair of Committee Manish Kumar Assistant Professor in Chemical Engineering Michael Janik Professor in Chemical Engineering Reka Albert Professor in Physics Phillip E. Savage Department Head and Graduate Program Chair Professor in Chemical Engineering ii ABSTRACT Nature relies on a wide range of enzymes with specific biocatalytic roles to carry out much of the chemistry needed to sustain life. Proteins catalyze the interconversion of a vast array of molecules with high specificity - from molecular nitrogen fixation to the synthesis of highly specialized hormones, quorum-sensing molecules, defend against disease causing foreign proteins, and maintain osmotic balance using transmembrane channel proteins. Ever increasing emphasis on renewable sources for energy and waste minimization has turned biocatalytic proteins (enzymes) into key industrial workhorses for targeted chemical conversions. Modern protein engineering is central to not only food and beverage manufacturing processes but are also often ingredients in countless consumer product formulations such as proteolytic enzymes in detergents and amylases and peptide-based therapeutics in the form of designed antibodies to combat neurodegenerative diseases (such as Parkinson’s, Alzheimer’s) and outbreaks of Zika and Ebola virus. However, successful protein design or tweaking an existing protein for a desired functionality has remained a constant challenge. This is mainly because of the complex energy landscape of proteins have not been fully discerned. Any amino acid change to a protein can bring about large-scale conformational changes that destabilize the whole structure. However, with the ease of availability of computing power, Monte Carlo based random sampling of the amino sequence space has become more amenable and has led to the discovery of several de novo protein sequences that fold like natural analogues. This thesis presents three new computational protein design tools targeted at redesign of – (a) channel proteins- for tunable pore size and chemistry to control the passage/ rejection of desired solutes in a membrane-separation module, (b) enzymes – by allowing prediction of amino acid insertions and deletions, along with substitutions to emulating natural evolution of proteins to switch their specificity towards/ from an intended substrate or cofactor, iii and (c) humanized antibody sequences that would possess structural complementarity to a disease causing antigen protein, and thereby neutralize it. All these tools employ a mixed-integer linear optimization approach for designing the optimal protein sequence, a molecular-mechanics force- field calculations (using CHARMM, and Rosetta packages) to assess the respective structures, and an iterative workflow that uses a Metropolis criterion to retain or reject the predicted designs and improve upon them. First, PoreDesigner provides a systematic approach to tune the pore size and inner pore wall chemistry of any channel protein which has potential applications from membrane- based separation of aqueous solutes to DNA sequencing. Next, we have developed an iterative enzyme redesign and optimization tool that enables the user to find active site mutations on the enzyme to alter substrate and cofactor specificity. Finally, our third tool is aimed at assembling fragments of human antibody using a mixed-integer linear optimization approach to obtain a complete a library of antibody variable domains that bind to a user-provided disease-causing antigen protein with high specificity. Binding scores are computed using non-bonded enthalpic energy models comprising harmonic potential terms for – electrostatics, solvation, hydrogen bonding, and van der Waal’s interaction. iv TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................................................... vi LIST OF TABLES ............................................................................................................................................. viii NOMENCLATURE .......................................................................................................................................... ix ACKNOWLEDGEMENTS .............................................................................................................................. x 1. Chapter 1 POREDESIGNER FOR TUNING SOLUTE SELECTIVITY IN A ROBUST AND HIGHLY PERMEABLE OUTER MEMBRANE PORE ........................................................................................................................................................................... 1 1.1 Significance .................................................................................................................................................. 1 1.2 Introduction .................................................................................................................................................. 2 1.3 Results .......................................................................................................................................................... 7 1.4 Discussion .................................................................................................................................................... 23 1.5 Materials and Methods ................................................................................................................................. 26 1.6 References .................................................................................................................................................... 36 2. Chapter 2 BRIEF HISTORY OF ENZYME REDESIGN FROM DIRECTED EVOLUTION TO COMPUTATIONAL ENZYME REDESIGN ........................................................................................................................................................................... 43 2.1 Background .................................................................................................................................................. 43 2.2 Successes ...................................................................................................................................................... 56 2.3 New approaches and future directions ......................................................................................................... 69 2.4 References .................................................................................................................................................... 70 3. Chapter 3 IPRO+/- COMPUTATIONAL PROTEIN DESIGN TOOL ALLOWING FOR INSERTIONS AND DELETIONS ........................................................................................................................................................................... 85 3.1 Significance .................................................................................................................................................. 85 3.2 Introduction .................................................................................................................................................. 86 3.3 Materials and Methods ................................................................................................................................. 92 3.4 Results and Discussion ................................................................................................................................. 97 3.6 References .................................................................................................................................................... 111 4. Chapter 4 OPTMAVEN-2.0 FOR DE NOVO DESIGN OF VARIABLE ANTIBODY REGIONS AGAINST TARGETED ANTIGEN EPITOPES ........................................................................................................................................................................... 116 4.1 Significance .................................................................................................................................................. 116 4.2 Introduction .................................................................................................................................................. 118 4.3 Materials and Methods ................................................................................................................................. 120 4.4 Results .......................................................................................................................................................... 141 4.5 Summary and Discussion ............................................................................................................................. 154 4.6 References .................................................................................................................................................... 156 5. Chapter 5 SYNOPSIS ............................................................................................................................... 163 v LIST OF FIGURES