The Mechanism of the ATP- Independent Periplasmic Chaperone Sura in Outer Membrane Protein Biogenesis
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The mechanism of the ATP- independent periplasmic chaperone SurA in outer membrane protein biogenesis Julia Rose Humes Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences September 2018 Intellectual Property and Publication Statement The candidate confirms that the work submitted is her own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. Throughout this thesis the work directly attributable to the candidate is as follows: (i) Literature research and compilation of the document. (ii) The candidate performed all the experimental work and data analysis unless otherwise stated. Chapters 3 contain work from the following publication: Schiffrin, B. Calabrese A.N., Higgins A.J., Humes J.R., Ashcroft A.E., Kalli A.C., Brockwell D.J., Radford S.E.. Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding. J Mol Biol 429, 3776-3792 (2017). In this publication B. Schiffrin designed and performed the kinetic experiments, computer modelling and molecular dynamics simulations. A. N. Calabrese designed and performed the mass-spectrometry experiments. A Higgins carried out crosslinking and kinetics. J Humes carried out the binding experiments. A Kalli, D Brockwell, A Ashcroft and S Radford provided experimental advice and helped with manuscript preparation. ii Acknowledgments Firstly, I would like to thank my supervisors Prof Sheena Radford and Dr David Brockwell who have been guided me through the last four years and pushed me to achieve things I didn’t think I could. Thanks to the University of Leeds and the NIH for funding this project. I would also like to thank Dr Iain Manfeild, who trained me and allowed me to use the equipment in his lab to study the many molecular interactions in this thesis. Although my time in the NMR facility was brief, I learned so much and the help from Dr Arnout Kalverda has been invaluable. The struggles I’ve have encountered during my research have been lessened by the wonderful people I’ve worked with in the Radford lab. There is always a friendly face to give advice on why an experiment hasn’t worked and to go and have a drink with to help ease any science worries. In particular Nasir Khan our lab manager who always provides a smile and often delicious curries and really helped me settle into the lab all those years ago. Thanks to Jess Ebo and Matt Jackson who arranged so many social events to help turn work place proximity colleges into really good friends, as well as always having the kettle boiling when you needed a chat. Within the Radford empire, the small group which took me in and who I worked with most were the OMPire headed up by Bob Schiffrin who trained my up in the lab when I didn’t know which end of the pipette was which and has always come up with great suggestions and advice on my project. Anna Higgins, Jim Horne, Anton Calabrese, Paul White and Matt Iadanza who make up the OMPire are such a great group to work in and always willing to lend a hand as well as many terrible BAM jokes. I need to give special thanks to Anna Higgins, Robb Hollis and Amy Swanston who have been there for me both in the lab and out to share triumphs and defeats with a large glass of wine. iii My family have always been a constant support in everything I have achieved and they have always been there to cheer me on. My Mam and Dad have given me all the love I could ever need and many Sunday lunches to get me through. My big sister and my best friend Hannah, is always on the end of the phone through all my dramas she always knows what I need to hear. Finally, a huge thank you to Dr Zak Mitchell who is the most supportive boyfriend I could ask for and has taken care of me through my turbulent Yorkshire Marathon training, the ups and downs of my research and the stress of thesis writing, I don’t know what I would have done without him. iv Abstract Outer membrane proteins (OMPs) of Gram-negative bacteria travel from their site of synthesis in the cytoplasm, across the inner membrane and through the periplasm to the outer membrane (OM) prior to their folding to a functional form. To protect OMPs from misfolding or aggregation while traversing the periplasm a network of chaperones are employed. The OMPs must then reach the essential β-barrel assembly machinery (BAM) complex, which is involved in inserting OMPs into the OM. This process occurs in the absence of chemical energy as the periplasm is devoid of ATP and in a highly dynamic environment as the ‘leaky’ OM allows small molecules (<600 Da) to enter from the extracellular milieu. The major periplasmic chaperone for OMPs, SurA, is known to interact with a number of substrates and has been crosslinked to the BAM complex in vivo. SurA is composed of four domains, an N-terminal domain, two peptidyl-prolyl isomerase (PPIase) domains and a short C-terminal helical domain. In this work wild-type E. coli SurA and SurA truncation variants lacking one (P2) or both (N-Ct) of the PPIase domains have been studied. Using microscale thermophoresis, light scattering, native mass spectrometry and other biophysical techniques how each domain is involved in OMP binding, chaperoning and delivery to BAM is investigated. The results demonstrate that SurA binds unfolded OMPs, tOmpA and OmpT with μM affinity, agreeing with previous findings. The core domain (SurA N-Ct) is sufficient for this interaction, but the addition of the PPIase domains leads to a tighter binding. Light scattering experiments shows that SurA WT can prevent aggregation of the two model OMPs, but the removal of the PPIase domains reduces the chaperoning ability for the larger, more aggregation-prone OMP, OmpT. These observations demonstrate that the acquisition of the PPIase domains is advantageous for both OMP binding and chaperoning. An interaction between SurA and the BAM complex is also observed for the first time in vitro. Overall, the results reveal new insights into v how SurA binds and chaperones OMPs before delivering them to the BAM complex for folding in the OM. vi Table of Contents Intellectual Property and Publication Statement ........................................ ii Acknowledgments .................................................................................... iii Abstract .................................................................................................... v Table of Contents .................................................................................... vii List of Figures ........................................................................................... xi List of Tables .......................................................................................... xiv List of Equations ...................................................................................... xv Abbreviations ......................................................................................... xvi Chapter 1 Introduction ................................................................................ 1 1.1 Protein Folding and Proteostasis ..................................................... 1 1.2 Gram-negative bacteria ................................................................... 3 1.3 The Periplasm .................................................................................. 5 1.4 Outer Membrane Proteins (OMPs) .................................................. 6 1.5 Outer membrane protein translocation ............................................ 9 1.6 The BAM complex ......................................................................... 13 1.7 Protein Folding of Bacterial Proteins .............................................. 16 1.7.1 Cytoplasmic Protein Folding ................................................. 16 1.7.2 Protein Folding in the Bacterial Periplasm ............................ 18 1.8 Periplasmic Chaperones of Outer Membrane Proteins (OMPs) .... 22 1.8.1 Skp ....................................................................................... 22 1.8.2 FkpA ..................................................................................... 25 1.8.3 DegP .................................................................................... 27 1.8.4 SurA ..................................................................................... 28 1.9 Periplasmic stress responses ........................................................ 34 1.10 Peptiylprolyl Isomerases (PPIases) in protein folding .................... 36 1.11 Current Questions in the Field and Aims of this Thesis ................. 39 Chapter 2 Methods .................................................................................... 41 2.1 Materials and Reagents ................................................................. 41 2.1.1 General Chemicals ............................................................... 41 2.1.2 Molecular Biology Materials .................................................. 41 2.1.3 Protein Chemistry Materials ................................................. 42 2.2 Molecular Biology .........................................................................