DOCSLIB.ORG

Exploring the Sequence Landscape of the Model Protein Rop to Gain Insights Into Sequence-Stability Relationship in Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Exploring the Sequence Landscape of the Model Protein Rop to Gain Insights Into Sequence-Stability Relationship in Proteins Exploring the sequence landscape of the model protein Rop to gain insights into sequence-stability relationship in proteins DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nishanthi Panneerselvam Graduate Program in Biophysics The Ohio State University 2017 Dissertation Committee: Professor Thomas J. Magliery, Advisor Professor Charles E. Bell Professor Ralf A. Bundschuh Copyrighted by Nishanthi Panneerselvam 2017 Abstract Surface residues and surface electrostatics play an active role in maintaining protein stability. A combinatorial library randomizing five surface positions in Rop to NNK (K=G or T) codons was constructed. Interestingly, the consensus sequence had positively charged residues instead of negatively charged residues present in wild-type. Specifically, lysines were found more than arginines. To delve into this, all these five positions were individually mutated to Lys and Arg and four poly mutants were made by mutating to multiple lysines and arginines. Most of the point mutants were found to be active by Rop screen. All mutants were well folded as seen by circular dichroism studies. An important result was that most of the mutants were more thermally stable than the Cys-free wild-type scaffold AV-Rop. Thermodynamic parameters were found using Gibbs-Helmholtz analysis and the entropic change was higher for most mutants. Solubility was affected more in the poly mutants than in the point mutants. HSQC on selected variants revealed that the least stable mutant and poly mutants had more shifted peaks compared to the most stable mutant. No considerable differences were found between Lys and Arg mutants. Poly mutants had varied effects on activity and solubility compared to point mutants. ii If all the possible point mutants in a protein are subject to moderate selection for activity or function, high-throughput sequencing can measure the relative abundance of mutants in a bulk competition experiment which in turn gives the relative fitness of each mutant. This project involves making all possible point mutants of Rop by constructing 20 member positional libraries individually in 62 positions of Rop. Preliminary data includes proof-of-principle selection experiments in three positions of Rop (F14, D30 and D43). Variants were enriched 1,000 fold for activity by performing six rounds of growth based selection. Likewise, we are in the process of constructing 62 positional libraries which will be combined together in the selection screen, subject to enrichment and then sequenced by high-throughput methods to give the sequence landscape. Analyzing the sequence/fitness landscape of Rop will yield each mutant’s competitive advantage/disadvantage. iii Acknowledgments First, I would like to thank my advisor Dr. Thomas Magliery for providing me the opportunity to work in his lab. I have learnt a great deal from him and I have always admired his passion towards research. The degree wouldn’t have been possible if not for the valuable inputs from his expertise. I thank him for his constant support and encouragement during tough times. I owe him a lot for all this support. I thank the Magliery group members for providing support and guidance. I would like to thank Dr. Ralf Bundschuh for being on my committee and for advising me as the Director of Biophysics. I would also like to thank my committee members Dr. Chuck Bell and Dr. Christopher Jaroniec for providing me feedback and guidance. I would like to thank everyone else who supported me in some way through my graduate school career. I would like to thank my mom and dad who share a big part in finishing the degree by being there for me. I would also like to thank my bestie Geethi for every small and big help that she did to me. I want to thank my friends Kanu, Mithila, Aishu and Nitya for making graduate school fun. I want to thank my husband Karthik for the support during completion of the degree. I would like to thank my sisters for always being there for me. I want to thank my all other friends and family. iv Vita 2009................................................................B. Tech. Industrial Biotechnology, Anna University 2013................................................................M.S. Biophysics, The Ohio State University 2013-present ...................................................Graduate associate, Biophysics program, The Ohio State University Fields of Study Major Field: Biophysics v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi Chapter 1: Introduction ....................................................................................................... 1 1.1 Protein stability ......................................................................................................... 2 1.2 Plasmid regulation by the protein ‘ROP’ .................................................................. 5 1.3 ROP as a model protein ............................................................................................. 9 1.4 Role of surface residues in protein stability ............................................................ 15 1.5 Effect of surface electrostatics on protein stability ................................................. 18 1.6 Other factors that affect protein stability ................................................................. 23 1.7 Lysines vs Arginines on the protein surface ........................................................... 24 vi 1.8 Solubility ................................................................................................................. 27 1.9 Deep sequencing ..................................................................................................... 32 1.9.1 DNA Sequencing .............................................................................................. 34 1.9.2 Chain terminator sequencing ............................................................................ 35 1.9.3 Second generation sequencing.......................................................................... 39 1.9.4 Next generation sequencing methods ............................................................... 39 1.10 General applications of next generation sequencing ............................................. 52 1.11 Applications of deep sequencing in protein stability and fitness studies .............. 53 Chapter 2: Engineering surface residues of the four helix bundle protein Rop ................ 63 2.1 Summary ................................................................................................................. 64 2.2.1 Plasmids and strains.......................................................................................... 65 2.2.2 Rop NNK5-surface library construction ........................................................... 65 2.2.3 Screen and selection for active variants ........................................................... 66 2.2.4. Resequencing of pAC surface library .............................................................. 66 2.3 Results ..................................................................................................................... 69 2.4 Discussion ............................................................................................................... 78 vii Chapter 3: Effect of surface lysines and arginines on stability and other characteristics of Rop .................................................................................................................................... 80 3.1: Summary ................................................................................................................ 81 3.2 Materials and Methods ............................................................................................ 82 3.2.1 Cloning and Sequencing ................................................................................... 82 3.2.2 Solubility .......................................................................................................... 88 3.2.3 Activity ............................................................................................................. 88 3.2.4 Protein expression and purification .................................................................. 89 3.2.5 Circular Dichroism ........................................................................................... 91 3.2.6 Gibbs-Helmholtz Analysis ............................................................................... 91 3.2.7 HSQC NMR ..................................................................................................... 92 3.3 Results ..................................................................................................................... 93 3.3.1 Construction of lysine and arginine variants ...................................................
Load more

Recommended publications
  • Cyclotides Evolve
    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 218 Cyclotides evolve Studies on their natural distribution, structural diversity, and activity SUNGKYU PARK ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 ISBN 978-91-554-9604-3 UPPSALA urn:nbn:se:uu:diva-292668 2016 Dissertation presented at Uppsala University to be publicly examined in B/C4:301, BMC, Husargatan 3, Uppsala, Friday, 10 June 2016 at 09:00 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Faculty examiner: Professor Mohamed Marahiel (Philipps-Universität Marburg). Abstract Park, S. 2016. Cyclotides evolve. Studies on their natural distribution, structural diversity, and activity. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 218. 71 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9604-3. The cyclotides are a family of naturally occurring peptides characterized by cyclic cystine knot (CCK) structural motif, which comprises a cyclic head-to-tail backbone featuring six conserved cysteine residues that form three disulfide bonds. This unique structural motif makes cyclotides exceptionally resistant to chemical, thermal and enzymatic degradation. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. The cyclotides found in plants exhibit considerable sequence and structural diversity, which can be linked to their evolutionary history and that of their host plants. To clarify the evolutionary link between sequence diversity and the distribution of individual cyclotides across the genus Viola, selected known cyclotides were classified using signature sequences within their precursor proteins. By mapping the classified sequences onto the phylogenetic system of Viola, we traced the flow of cyclotide genes over evolutionary history and were able to estimate the prevalence of cyclotides in this genus.
    [Show full text]
  • Of Modified Peptides 660 Absorption
    Index A Aequorea victoria 182, 192 post-column aerosols, danger of contamination 770 derivatization 303–305 Abbe, Ernst 181, 187, 485, 493 affinity capillary electrophoresis pre-column absolute molecule mass 4 (ACE) 285–286 derivatization 305–308 absolute quantification (AQUA) of binding constant, determined by 285 reagents used for 310 modified peptides 660 changing mobility 286 sample preparation 302 absorption complexation of monovalent acidic hydrolysis 302 bands, of most biological molecules 139 protein–ligand complexes 285 alkaline hydrolysis 303 measurement 140–142 affinity chromatography 91, 268, 650 enzymatic hydrolysis 303 of photon 135 affinity purification mass spectrometry using mass spectrometry 309 spectroscopy 131 (AP-MS) 381, 1003 L-α-amino acid residues/termini 225 acetic acid 228 agarose concentrations amino acid sequence analysis acetonitrile 227 DNA fragments, coarse separation milestones in 319 N-acetyl-α-D-glucosamine of 692 problems 322 (αGlcNAc) 579 migration distance and fragment amino acids 323–324 acetylated proteins length 692 background 324 detection of 651 agarose gels, advantages of 260 initial yield 324 separation and enrichment 649 agglutination 72 modified amino acids 324 acetylation 224, 645–647 aggregation number 288 purity of chemicals 324 sites, in proteins 656 AK2-antibodies 102 sample to be sequenced 322–323 identification of 655 alanine 562 sensitivity of HPLC system 325 N-acetyl-β-D-glucosamine alanine-scanning method 870 state of the art 325 (βGlcNAc) 579 albumin 3, 995 6-aminoquinoyl-N-hydroxysuccinimidyl
    [Show full text]
  • Nonproteinogenic Deep Mutational Scanning of Linear and Cyclic Peptides
    Nonproteinogenic deep mutational scanning of linear and cyclic peptides Joseph M. Rogersa, Toby Passiouraa, and Hiroaki Sugaa,b,1 aDepartment of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; and bCore Research for Evolutionary Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan Edited by David Baker, University of Washington, Seattle, WA, and approved September 18, 2018 (received for review June 10, 2018) High-resolution structure–activity analysis of polypeptides re- mutants that can be constructed (18). Moreover, it is possible to quires amino acid structures that are not present in the universal combine parallel peptide synthesis with measures of function genetic code. Examination of peptide and protein interactions (19). However, these approaches cannot construct peptide li- with this resolution has been limited by the need to individually braries with the sequence length and numbers that deep muta- synthesize and test peptides containing nonproteinogenic amino tional scanning can, which, at its core, uses high-fidelity nucleic acids. We describe a method to scan entire peptide sequences with acid-directed synthesis of polypeptides by the ribosome. multiple nonproteinogenic amino acids and, in parallel, determine Ribosomal synthesis (i.e., translation) can be manipulated to the thermodynamics of binding to a partner protein. By coupling include nonproteinogenic amino acids (20). In vitro genetic code genetic code reprogramming to deep mutational scanning, any reprogramming is particularly versatile, allowing for the in- number of amino acids can be exhaustively substituted into pep- corporation of amino acids with diverse chemical structures (21). tides, and single experiments can return all free energy changes of Flexizymes, flexible tRNA-acylation ribozymes, can load almost binding.
    [Show full text]
  • Computational Prediction of Alanine Scanning and Ligand Binding Energetics in G-Protein Coupled Receptors
    Computational Prediction of Alanine Scanning and Ligand Binding Energetics in G-Protein Coupled Receptors Lars Boukharta, Hugo Gutie´rrez-de-Tera´n, Johan A˚ qvist* Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Uppsala, Sweden Abstract Site-directed mutagenesis combined with binding affinity measurements is widely used to probe the nature of ligand interactions with GPCRs. Such experiments, as well as structure-activity relationships for series of ligands, are usually interpreted with computationally derived models of ligand binding modes. However, systematic approaches for accurate calculations of the corresponding binding free energies are still lacking. Here, we report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 receptor and series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Citation: Boukharta L, Gutie´rrez-de-Tera´nH,A˚qvist J (2014) Computational Prediction of Alanine Scanning and Ligand Binding Energetics in G-Protein Coupled Receptors. PLoS Comput Biol 10(4): e1003585. doi:10.1371/journal.pcbi.1003585 Editor: Alexander Donald MacKerell, University of Maryland, Baltimore, United States of America Received February 7, 2014; Accepted March 12, 2014; Published April 17, 2014 Copyright: ß 2014 Boukharta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    [Show full text]
  • High-Throughput Mutagenesis Using a Two-Fragment PCR Approach
    Research Collection Journal Article High-throughput mutagenesis using a two-fragment PCR approach Author(s): Heydenreich, Franziska M.; Miljuš, Tamara; Jaussi, Rolf; Benoit, Roger; Milić, Dalibor; Veprintsev, Dmitry B. Publication Date: 2017-07 Permanent Link: https://doi.org/10.3929/ethz-b-000191258 Originally published in: Scientific Reports 7, http://doi.org/10.1038/s41598-017-07010-4 Rights / License: Creative Commons Attribution 4.0 International This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library www.nature.com/scientificreports OPEN High-throughput mutagenesis using a two-fragment PCR approach Received: 17 March 2017 Franziska M. Heydenreich 1,2, Tamara Miljuš 1,2, Rolf Jaussi1, Roger Benoit1, Accepted: 20 June 2017 Dalibor Milić 1,3 & Dmitry B. Veprintsev1,2 Published: 28 July 2017 Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efciency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efciency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that signifcantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of resulting fragments.
    [Show full text]
  • Download Author Version (PDF)
    Chemical Science Accepted Manuscript This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/chemicalscience Page 1 of 7 Chemical Science Chemical Science RSCPublishing EDGE ARTICLE Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids† Cite this: DOI: 10.1039/x0xx00000x Ivana Drienovská, Ana Rioz-Martínez, Apparao Draksharapu and Gerard Roelfes* Artificial metalloenzymes have emerged as an attractive new approach to enantioselective Received 00th January 2012, Accepted 00th January 2012 catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal- DOI: 10.1039/x0xx00000x binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asymmetric Friedel-Crafts alkylation reactions and up to 83% ee for the product was www.rsc.org/chemicalscience achieved.
    [Show full text]
  • Directed Evolution of Stabilized Peptides with Bacterial Display
    Directed Evolution of Stabilized Peptides with Bacterial Display Tejas A. Navaratna A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Engineering) in the University of Michigan 2020 Doctoral Committee: Associate Professor Greg Thurber, Chair Professor James Bardwell Professor Erdogan Gulari Associate Professor Xiaoxia Nina Lin Associate Professor Fei Wen Tejas Navaratna [email protected] ORCID iD: 0000-0003-2976-0137 © Tejas Navaratna 2020 Acknowledgments I deeply thank Professor Greg Thurber for his guidance, trust, and support in me. I cannot count the number of times I came into his office unsure of future research paths and came away inspired and eager to probe new directions. He gave me the opportunity and freedom to explore my ideas, while also being an excellent mentor and advisor in this journey. I also thank my other committee members, Professors Jim Bardwell, Erdogan Gulari, Nina Lin, and Fei Wen for their support and productive discussions. I also would like to thank Professor Jim Van Deventer, Professor Cathy Fromen, Professor Kevin Solomon, and Dr. Marco Jost for helpful career advice. I would also like to acknowledge Andrew Min and Liz Goulston, two undergraduate students I have had the pleasure of mentoring. Working with them led me to realize my passion for mentorship. Other Thurber Lab members that I closely worked with are Lydia Atangcho, Liang Zhang, Marshall Case, and Mukesh Mahajan – I am deeply grateful for your advice and support. Finally, I would also like to thank Sumit Bhatnagar, Cornelius Cilliers, Bruna Menezes, Eshita Khera, Ian Nessler, Reggie Evans, Anna Kopp, Celia Dong, Haolong Huang, and Yinuo Chen for fostering a social, friendly, and wonderfully collaborative lab environment, even through the COVID-19 shutdown– I couldn’t have asked for better labmates.
    [Show full text]
  • The Optimized Method for Chemical Synthesis of Cyclotide Precursor and the Prediction of the Function of Cyclotide Prodomain in Biosynthesis of Cyclic Protein
    The optimized method for chemical synthesis of cyclotide precursor and the prediction of the function of cyclotide prodomain in biosynthesis of cyclic protein Park SungKyu Degree project in applied biotechnology, Master of Science (2 years), 2009 Examensarbete i tillämpad bioteknik 45 hp till masterexamen, 2009 Biology Education Centre and Department of Medicinal Chemistry at Division of Pharmacognosy, Uppsala University Supervisors: Assistant Professor Dr. Ulf Goransson and Dr. Sunithi Gunasekera Abstract Cyclotides are mini-proteins of approximately 30 amino acids residues that have a unique structure consisting of a head-to-tail cyclic backbone with a knotted arrangement of three disulfide bonds. This unique structure uniqueness gives exceptional stability to chemical, enzymatic and thermal treatments. Cyclotides display various bioactivities, such as anti-HIV, uterotonic, cytotoxic, and insecticidal activity. Due to the structural stability and their wide range of bioactivity, cyclotides have been implicated as ideal drug scaffolds and for development into agricultural and biotechnological agents. To date the exact mechanism by which the complex knotted topology of cyclotides is formed, is the biosynthesis of cyclotides, remains largely unknown. Certain catalytic proteins are assumed to be involved in the processing of the linear cyclotide precursor into the mature circular protein. In order to elucidate the mechanism of the putative proteins, this project aims to synthesize the full length precursor of the prototypical cylcotide kalata B1. The present paper summarizes 1) the biosynthesis of cyclotides by a literature survey, 2) the anticipated roles of cyclotide prodomain in its native folding based on its predicted model structure, 3) the optimized methods for the formation of peptide α- thioester 4) the mechanism of native chemical ligation in the model peptides through thiol-thioester exchange.
    [Show full text]
  • The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis
    International Journal of Molecular Sciences Review The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis Vladimir Kubyshkin 1,* and Nediljko Budisa 1,2,* 1 Department of Chemistry, University of Manitoba, Dysart Rd. 144, Winnipeg, MB R3T 2N2, Canada 2 Department of Chemistry, Technical University of Berlin, Müller-Breslau-Str. 10, 10623 Berlin, Germany * Correspondence: [email protected] (V.K.); [email protected] or [email protected] (N.B.); Tel.: +1-204-474-9321 or +49-30-314-28821 (N.B.) Received: 24 September 2019; Accepted: 3 November 2019; Published: 5 November 2019 Abstract: A central question in the evolution of the modern translation machinery is the origin and chemical ethology of the amino acids prescribed by the genetic code. The RNA World hypothesis postulates that templated protein synthesis has emerged in the transition from RNA to the Protein World. The sequence of these events and principles behind the acquisition of amino acids to this process remain elusive. Here we describe a model for this process by following the scheme previously proposed by Hartman and Smith, which suggests gradual expansion of the coding space as GC–GCA–GCAU genetic code. We point out a correlation of this scheme with the hierarchy of the protein folding. The model follows the sequence of steps in the process of the amino acid recruitment and fits well with the co-evolution and coenzyme handle theories. While the starting set (GC-phase) was responsible for the nucleotide biosynthesis processes, in the second phase alanine-based amino acids (GCA-phase) were recruited from the core metabolism, thereby providing a standard secondary structure, the α-helix.
    [Show full text]
  • Using Large-Scale Mutagenesis to Guide Single Amino Acid Scanning Experiments
    bioRxiv preprint doi: https://doi.org/10.1101/140707; this version posted May 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Using large-scale mutagenesis to guide single amino acid scanning experiments Vanessa E. Gray1, Ronald J. Hause1, Douglas M. Fowler1,2 1Department of Genome Sciences, University of Washington, Seattle, WA, USA 2Department of Bioengineering, University of Washington, Seattle, WA, USA Correspondence to [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/140707; this version posted May 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Abstract 2 Alanine scanning mutagenesis is a widely-used method for identifying protein positions 3 that are important for function or ligand binding. Alanine was chosen because it is 4 physicochemically innocuous and constitutes a deletion of the side chain at the β- 5 carbon. Alanine is also thought to best represent the effects of other mutations; 6 however, this assumption has not been formally tested. To determine whether alanine 7 substitutions are always the best choice, we analyzed 34,373 mutations in fourteen 8 proteins whose effects were measured using large-scale mutagenesis approaches. We 9 found that several substitutions, including histidine and asparagine, are better at 10 recapitulating the effects of other substitutions.
    [Show full text]
  • Rapid Mapping of Protein Functional Epitopes by Combinatorial Alanine Scanning
    Rapid mapping of protein functional epitopes by combinatorial alanine scanning Gregory A. Weiss*, Colin K. Watanabe†, Alan Zhong‡, Audrey Goddard‡, and Sachdev S. Sidhu*§ Departments of *Protein Engineering, †Bioinformatics, and ‡Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080 Communicated by James A. Wells, Sunesis Pharmaceuticals, Inc., Redwood City, CA, June 1, 2000 (received for review March 2, 2000) A combinatorial alanine-scanning strategy was used to determine Herein, we describe shotgun scanning, a general combinato- simultaneously the functional contributions of 19 side chains rial method for mapping functional epitopes of proteins. Shotgun buried at the interface between human growth hormone and the scanning combines the concepts of alanine-scanning mutagen- extracellular domain of its receptor. A phage-displayed protein esis (13–15) and binomial mutagenesis (16) with phage display library was constructed in which the 19 side chains were prefer- technology (17, 18). The method is extremely rapid, because entially allowed to vary only as the wild type or alanine. The library many side chains are analyzed simultaneously, and the need for pool was subjected to binding selections to isolate functional protein purification and biophysical analysis is circumvented. ͞ clones, and DNA sequencing was used to determine the alanine Instead, the energetic contributions of individual side chains are wild-type ratio at each varied position. This ratio was used to derived from statistical analysis of DNA sequences. We used calculate the effect of each alanine substitution as a change in free shotgun scanning to study the binding interaction between energy relative to that of wild type.
    [Show full text]
  • Computational Methods to Identify and Target Druggable Binding Sites at Protein-Protein Interactions in the Human Proteome
    COMPUTATIONAL METHODS TO IDENTIFY AND TARGET DRUGGABLE BINDING SITES AT PROTEIN-PROTEIN INTERACTIONS IN THE HUMAN PROTEOME David Xu Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the School of Informatics and Computing, Indiana University September 2019 Accepted by the Graduate Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Doctoral Committee ______________________________________ Huanmei Wu, PhD, Co-Chair ______________________________________ Samy Meroueh, PhD, Co-Chair April 15, 2019 ______________________________________ Xiaowen Liu, PhD ______________________________________ Sarath Chandra Janga, PhD ______________________________________ Yunlong Liu, PhD ii © 2019 David Xu iii DEDICATION To my family, without whom this journey would not be possible. iv ACKNOWLEDGEMENT First and foremost, I would like to express my sincere appreciation to my advisor Dr. Samy Meroueh for his support during my graduate studies and related research over the past years. I would also like to thank him for his insight, generosity, and support throughout this experience, without whom this would not be possible. Sincere gratitude is given to my committee co-chair, Dr. Huanmei Wu, for her time and guidance through the Ph.D. process. I would also like to thank all my committee members, Dr. Xiaowen Liu, Dr. Sarath Chandra Janga, and Dr. Yunlong Liu, for their valuable comments and suggestions on my work. I would like to express my sincerest gratitude to all my colleagues through the years in the Center for Computational Biology and Bioinformatics: Dr. Liwei Li, Dr. Yubing Si, Dr. Michael Wu, Dr.
    [Show full text]