i Micelles and bicelles as membrane mimics for membrane protein investigations Ashton Tirrell Brock Columbia, South Carolina B.S. Chemistry, Winthrop University, 2011 A Dissertation presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Department of Chemistry University of Virginia May, 2016 ii © Copyright by Ashton Tirrell Brock All rights reserved. May 2016. iii “Call it a clan, call it a network, call it a tribe, call it a family. Whatever you call it, whoever you are, you need one.” – Jane Howard (1935-1996) US journalist, writer “Nothing will work unless you do.” – Maya Angelou (1928-2014) US poet, author, civil rights activist iv Abstract Membrane proteins have essential cellular functions in transport, signaling, energy conversion and catalysis. These ubiquitous proteins comprise approximately 30% of the genome, and with their roles in intra- and extracellular communication, membrane proteins are the target of over 60% of the therapeutic drugs on the market. While membrane protein research is imperative, there is significantly less known about the function, stability, and structure of these proteins as compared to soluble proteins. Membrane protein research is challenged by the native environment of these proteins: the lipid bilayer. Bilayer mimics such as detergent micelles and lipid-detergent bicelles are often used to isolate membrane proteins. Utilizing these mimics requires an extensive screening process to identify the appropriate amphiphilic environment for biophysical characterization, functional assays, and high-resolution structure determination of membrane proteins. To enable rational mimic selection, the goal of this research is to investigate the structural properties of micelles and bicelles to determine how their physical characteristics can forward membrane protein research. Often considered more membrane-like than micelles, the “ideal” bicelle is composed of a lipid bilayer encapsulated by a detergent rim. Because of the propensity for detergents to form ellipsoid micelles in aqueous environments, the feasibility of distinctly separated lipid and detergent domains suggested by the “ideal” bicelle model is questioned, especially in respect to detergent-rich bicelles. To investigate the viability of the “ideal” bicelle, the structure and composition of detergent-rich bicelles was elucidated with small angle X-ray and neutron scattering. Small angle scattering results suggest that detergent-rich bicelles form ellipsoid structures similar to mixed micelles, with higher v degrees of mixing between lipid and detergent components than suggested by the “ideal” model. As lipid concentration is increased, the aggregate transitions from a mixed micelle to bicelle structure. This research emphasizes the importance in understanding the structures of the mimics used for membrane protein research; the size and shape of these ellipsoidal detergent-rich bicelles can have a significant impact on mimic selection. The influence of detergent micelles on protein function was also investigated. Understanding the key physical detergent properties that impact protein activity will also guide membrane mimic selection. The activity of several membrane proteins is currently being assayed in varying detergent micelles to correlate trends between the micelle and active, stable protein. Functional results from two membrane enzymes, outer membrane phospholipase A1 (OMPLA) and lipoprotein signal peptidase A (LspA) suggest that the detergent head group charge and size has a major effect on protein activity, and the carbon tails of these detergents have minor impacts on detergent binding and catalysis. Identifying similar trends with other protein-detergent complexes will guide the selection of the appropriate detergent to use for membrane protein research, and ameliorate arduous detergent screening. vi Table of Contents Copyright page .................................................................................................................... II Dedication ..........................................................................................................................III Abstract ............................................................................................................................. IV Table of contents ............................................................................................................... VI List of Figures ..................................................................................................................... X List of Tables ................................................................................................................... XII Chapter 1: Membrane proteins in research ..........................................................................1 1.1 Membrane protein background ................................................................................1 1.2 Significance of membrane proteins .........................................................................6 1.3 The native lipid bilayer ............................................................................................7 1.3.1 Lipid bilayer assembly .................................................................................7 1.3.2 The fluid mosaic model ...............................................................................8 1.3.3 The bacterial inner membrane ...................................................................11 1.3.4 The bacterial outer membrane ...................................................................11 1.4 Functions of membrane proteins ............................................................................12 1.4.1 OMPLA structure and function .................................................................14 1.4.2 PagP structure and function .......................................................................19 1.4.3 OmpT structure and function .....................................................................24 1.4.4 LspA structure and function.......................................................................26 1.5 Thesis objectives ....................................................................................................32 1.6 References ..............................................................................................................33 Chapter 2: Membrane mimics ............................................................................................42 2.1 Detergent micelles .................................................................................................42 2.1.1 Self-association of detergent monomers ....................................................42 2.1.2 General detergent micelle categories .........................................................44 2.1.3 Micelle properties ......................................................................................47 2.1.3.1 Effects of head group .....................................................................47 2.1.3.2 Effects of alkyl tail .........................................................................51 vii 2.1.4 Solubilization and folding of membrane proteins in micelles ...................52 2.1.5 Mixed micelles ...........................................................................................54 2.2 Bicelles ...................................................................................................................55 2.2.1 Bicelle composition ...................................................................................58 2.3 Difficulties with protein-mimic investigations ......................................................60 2.3.1 Challenges with protein-detergent complexes ...........................................61 2.3.2 Challenges with low-q bicelle structures ...................................................63 2.4 References ..............................................................................................................65 Chapter 3: The influence of detergents on the function of β-barrel enzymes ....................70 3.1 Protein-detergent complex formation with OMPLA .............................................70 3.2 Pre-assay biophysical characterization of OMPLA ...............................................75 3.2.1 Detergent exchange verification and concentration determination with NMR ..........................................................................................................75 3.2.2 Determination of the OMPLA oligomeric state .........................................77 3.3 OMPLA activity assay design ...............................................................................83 3.4 Structural considerations for OMPLA activity ......................................................88 3.5 OMPLA activity in varying detergents ..................................................................88 3.5.1 Effects of detergent micelles on OMPLA substrate turnover rates ...........94 3.5.2 Effects of detergent micelles on OMPLA substrate affinity ......................96 3.5.3 Additional remarks of OMPLA activity ....................................................98 3.6 Future directions ..................................................................................................100 3.6.1 PagP purification and activity in detergent micelles................................100 3.6.2 OmpT purification and activity assay design ...........................................101 3.7