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Substrate Adaptability of Periplasmic Substrate-Binding Proteins As a Function of Their Evolutionary Histories" (2014)

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Substrate Adaptability of Periplasmic Substrate-Binding Proteins As a Function of Their Evolutionary Histories University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 8-27-2014 Substrate Adaptability of Periplasmic Substrate- binding Proteins as a Function of their Evolutionary Histories Nathalie Boucher University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Boucher, Nathalie, "Substrate Adaptability of Periplasmic Substrate-binding Proteins as a Function of their Evolutionary Histories" (2014). Doctoral Dissertations. 533. https://opencommons.uconn.edu/dissertations/533 β-gal Substrate Adaptability of Periplasmic Substrate-binding Proteins as a Function of their Evolutionary Histories Nathalie Boucher, Ph.D. University of Connecticut, 2014 The ATP-binding cassette (ABC) transporters play an important role in the uptake of nutrients in bacterial and archaeal cells. The bacterium Thermotoga maritima has an unusually high number of ABC transporters. However, only a few of them have been characterized due to the variety of substrates they can transport and the time consuming efforts needed to thoroughly characterize each ABC transporter. In this work, a technique has been optimized to determine the sugars that interact with the substrate- binding proteins (SBP) of the ABC transporter complexes in Thermotoga maritima. Using this high throughput assay, the ligands of trehalose-binding protein (TreE) and xylose-binding protein (XylE2) in Thermotoga maritima as well as representatives of the mannoside-binding proteins (Man) in the Thermotoga species were identified. The phylogeny of the mannose-binding proteins is interesting because of the presence of two paralogous proteins in this family (ManD and ManE). Ten representatives of the mannoside-binding proteins (ManD and ManE) encoded in the Thermotogales were characterized. At 37°C and 60°C, the ManD and ManE homologs bind cellobiose, cellotriose, cellotetraose, β-mannotriose, and β-mannotetraose. However, the ManE homologs have additional function, as they are able to bind β-mannobiose, laminaribiose, laminaritriose and sophorose. i Nathalie Boucher – University of Connecticut, 2014 An examination of the selective pressure that acted on manD and manE and the identification of the residues located in the binding-site of their encoded proteins suggest that early in the evolution of manD and manE, these genes had different codon sites under positive selection which encode important regions of their binding site. Taken together, these analysis suggest that these paralogs likely evolved under different evolutionary constrains. ii Substrate Adaptability of Periplasmic Substrate-binding Proteins as a Function of their Evolutionary Histories Nathalie Boucher B.S. Laval University, Quebec, Canada, 2000 M.Sc. Laval University, Quebec, Canada, 2002 Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy At the University of Connecticut 2014 iii Copyright by Nathalie Boucher 2014 iv 2014 APPROVAL PAGE Doctor of Philosophy Dissertation Substrate Adaptability of Periplasmic Substrate-binding Proteins as a Function of their Evolutionary Histories Presented by Nathalie Boucher, B.S., M.Sc. Major Advisor________________________________ Kenneth M. Noll Associate Advisor________________________________ Daniel J. Gage Associate Advisor________________________________ J. Peter Gogarten University of Connecticut 2014 ii Acknowledgements I would like to thank my advisor, Dr. Kenneth Noll, for his mentorship during my graduate studies. Dr. Noll welcomed me in his laboratory and helped me find the best project that matched my skills. I greatly appreciate his willingness to incorporate my scientific ideas and I am grateful for all his professional advice. I thank my labmates, especially Nicholas and Chaman. Together, we had numerous scientific and non-scientific debates that taught me other points of view on different topics. I thank Kunica for all her help while I was in Albany and Kristen for her helpful comments and edits on my thesis. I also thank my past laboratory members for their friendship during those years. I thank my family, especially Pascal for his unconditional support through my graduate studies. I am grateful to my son, at first, for all those toothless smiles, and later, for all those great smiles that lighted up my days. I dedicate this thesis to my aunt, who was a mother to me and who saw the potential in me. She encouraged me to achieve my dreams and she taught me to stand up for my ideas. iii Table of Contents Chapter 1 ........................................................................................................................... 1 General introduction ........................................................................................................ 1 Evolutionary history of the ABC transporters and the SBP ............................................ 4 The Thermotogales .......................................................................................................... 6 Carbohydrate ABC transporters in Thermotoga maritima .............................................. 7 Carbohydrate uptake: catabolism and regulation ............................................................. 8 The SBP and the substrate translocation ........................................................................ 11 Objective statement ........................................................................................................ 14 Chapter overviews .......................................................................................................... 15 Contribution from other researchers ............................................................................ 17 Chapter 2 ......................................................................................................................... 18 Identification of genomic variations of different genomovars of Thermotoga maritima ........................................................................................................................... 18 Introduction ..................................................................................................................... 18 Materials and Methods ................................................................................................... 21 Strains ............................................................................................................................ 21 PCR amplification and DNA sequencing ...................................................................... 21 Sequence depositions ..................................................................................................... 23 Prediction software ........................................................................................................ 23 Data acquisition and phylogenetic analysis ................................................................... 23 Results .............................................................................................................................. 27 Identification of two genomovars of Thermotoga maritima MSB8 .............................. 27 Sequence of the genomic DNA deleted from the genomovar TIGR ............................. 28 iv Evolution of Mal and Tre transporters ........................................................................... 33 Genetic variations of malF2........................................................................................... 34 Discussion......................................................................................................................... 36 Recent up-dates: sequencing project of T. maritima MSB8 genomovar DSM3109 ..... 36 Conclusion ....................................................................................................................... 39 Chapter 3 ......................................................................................................................... 41 Development of a screening assay to identify the ligands that interact with thermophilic substrate-binding proteins (SBPs) .......................................................... 41 Introduction ..................................................................................................................... 41 DSF assay as a screening method to detect ligand-binding interactions ....................... 42 Principle of the DSF assay ............................................................................................. 43 Materials and Methods ................................................................................................... 46 Differential scanning fluorimetry (DSF) screening of ligand binding ........................... 46 Sugar purity .................................................................................................................... 47 Results .............................................................................................................................. 48 Effect of the change of pH and ligand concentration on the unfolding temperature ..... 48 DSF analysis of ligand binding of MalE1 and MalE2 ................................................... 54 Discussion......................................................................................................................... 58 Adaptation of the DSF assay for use with thermophilic SBPs ...................................... 58 Conclusion ......................................................................................................................
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