UNIVERSITY OF CALIFORNIA, SAN DIEGO SAN DIEGO STATE UNIVERSITY Beta-Hairpin Fusion to Alpha-Helical Domains for Exploring Recombinant Protein Stability in E. coli A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemistry by Melissa E. Lokensgard Committee in Charge: University of California, San Diego Professor Elizabeth Komives Professor Ross Walker San Diego State University Professor John Love, Chair Professor Tom Huxford Professor Roland Wolkowicz 2016 Copyright Melissa E. Lokensgard, 2016 All Rights Reserved The Dissertation of Melissa E. Lokensgard is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair SIGNATURE PAGE University of California, San Diego San Diego State University 2016 iii DEDICATION For Matt, Mom, and Dad. But mostly for me. iv TABLE OF CONTENTS Signature Page .............................................................................................................. iii Dedication ..................................................................................................................... iv Table of Contents ............................................................................................................ v List of Figures ............................................................................................................. viii List of Tables .................................................................................................................. x Acknowledgements ....................................................................................................... xi Vita .............................................................................................................................. xiii Abstract of the Dissertation ........................................................................................ xiv Chapter 1: Introduction ................................................................................................... 1 1.2 Perspective .......................................................................................................... 2 1.3 Target Definition ................................................................................................. 3 1.4 Four Small Domains for Protein Design ............................................................. 4 1.4.1 Protein A .................................................................................................... 5 1.4.2 Protein G .................................................................................................... 6 1.4.3 Ubiquitin .................................................................................................... 8 1.4.4 Engrailed .................................................................................................... 9 1.5 References ......................................................................................................... 20 Chapter 2: Fusion of β-hairpins to α-helical domains enhances recombinant expression accumulation by reducing effectiveness of host degradation stress response .............. 22 2.1 Introduction ....................................................................................................... 23 2.2 Materials and Methods ...................................................................................... 25 2.2.1 Molecular Cloning, Expression, and Purification .................................... 25 v 2.2.2 Expression Quantification ........................................................................ 26 2.2.3 Circular Dichroism (CD) ......................................................................... 27 2.2.4 Microscale Thermophoresis (MST) ......................................................... 28 2.2.5 Nuclear Magnetic Resonance .................................................................. 29 2.3 Results ............................................................................................................... 33 2.3.1 Increased expression of helical constructs in BL21(DE3) cytosol with added b-hairpin sequence ................................................................................. 33 2.3.2 Helical domains with b-hairpins contain added b character .................... 37 2.3.3 Unfolding of helical domain is unaffected by added b-hairpins .............. 41 2.3.4 b-Hairpins influence interactions with E. coli ClpS ................................ 48 2.4 Discussion ......................................................................................................... 53 2.4.1 Small helical domains can be vulnerable in the E. coli cytosol ............... 53 2.4.2 b-Hairpin sequences from stable domains can act as miniature tags without affecting protein architecture or stability ............................................. 55 2.5 Conclusions ....................................................................................................... 57 2.6 Future Direction ................................................................................................ 57 2.6.1 Nuclear Magnetic Resonance .................................................................. 58 2.6.2 Full-length Fusions .................................................................................. 58 2.6.3 Further explorations of the Clp system .................................................... 60 2.7 Acknowledgment .............................................................................................. 61 2.8 References ......................................................................................................... 62 Chapter 3: Circular Dichroism Spectroscopy of Pyrrolopyrimidine Derivatives and Nucleic Acids ................................................................................................................ 66 vi 3.1 Use of Circular Dichroism Spectroscopy to Assign Configurations of Atropisomeric Pyrrolopyrimidine Derivatives ....................................................... 67 3.1.1 Introduction .............................................................................................. 67 3.1.2 Methods.................................................................................................... 68 3.1.3 Results and Discussion ............................................................................ 69 3.1.4 Conclusions .............................................................................................. 76 3.2 Circular Dichroism Spectroscopy for Thermodynamic Characterization of Novel Fluorescent Oligonucleotides ....................................................................... 77 3.2.1 Introduction .............................................................................................. 77 3.2.2 Methods.................................................................................................... 78 3.2.3 Results and Discussion ............................................................................ 78 3.2.4 Conclusions .............................................................................................. 87 3.4 References ......................................................................................................... 88 vii LIST OF FIGURES Figure 1.1 Staphylococcal Protein A (SpA) ................................................................. 11 Figure 1.2 SpA IgG Fc contacts .................................................................................... 12 Figure 1.3 SpA and SpA(I17W) models ....................................................................... 13 Figure 1.4 Streptococcal Protein G ............................................................................... 14 Figure 1.5 Gβ1 IgG Fc contacts .................................................................................... 15 Figure 1.6 Primary amino acid sequences of Love Lab Gβ1 variants .......................... 16 Figure 1.7 Backbone alignment of Ub and Gβ1 domains ............................................. 17 Figure 1.8 En homeodomain DNA contacts ................................................................. 18 Figure 1.9 En destabilized variants I41V and I41A ...................................................... 19 Figure 2.1 Primary Amino Acid Sequences of Constructs ........................................... 31 Figure 2.2 Sequences and secondary structures of parent domains .............................. 32 Figure 2.3 Expression levels of β-hairpin fusions to SpA ............................................ 34 Figure 2.4 Expression levels of β-hairpin fusions to En ............................................... 35 Figure 2.5 Expression levels of destabilized variants ................................................... 36 Figure 2.6 Circular Dichroism spectra of SpA variants ................................................ 38 Figure 2.7 Circular Dichroism spectra of En variants .................................................. 39 Figure 2.8 Thermal denaturation curves of SpA variants. ............................................ 42 Figure 2.9 Thermal denaturation curves of En variants ................................................ 43 Figure 2.10 Spectral response to temperature of SpA variants ..................................... 45 Figure 2.11 15N-1H HSQC spectrum of SpA-NH. ........................................................ 46 Figure 2.12 15N-1H HSQC spectrum of NH-SpA. ........................................................ 47 Figure 2.13 Raw MST