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Developing Nanodiscs As a Tool for Low Resolution Studies Of FACU LTY OF SCIENCE UNIV ERSI TY O F C O PENHAGE N PhD thesis Nicholas Skar-Gislinge Developing Nanodiscs as a Tool for Low Resolution Studies of Membrane Proteins Ac ademic advisor: Lise Arleth Submitted: 28/02/2014 ii Abstract Phospholipid nanodiscs are 10 nm sized disc shaped particles consisting of roughly 150 phos- pholipids arranged in a central⇠ bilayer, stabilized by two amphipathic protein ”belts” that wrap around the rim of the bilayer. Because they contain a small bilayer leaflet, in which membrane proteins can be incorporated, they can be used as a tool for solution studies of membrane pro- teins. So far most of the studies of membrane proteins using nanodiscs have been concerning the function of the incorporated membrane protein. However, due to the good control of the size and lipid composition of the nanodisc system, they seem an ideal tool for expanding the use of small angle scattering from studying water soluble proteins, to also include membrane proteins inserted into nanodiscs. This has been the main goal of this thesis. In order to reach this goal it is necessary to have a detailed understanding of the nanodisc system, without the membrane protein, as well at the self-assembly process in general, and in particular in relation to incorporation of membrane proteins. This was the aim of work done early in this thesis. Here a detailed model for the small angle x-ray and neutron scattering from the empty nanodisc system was derived and used to describe the nanodisc system with great detail. The high level of detail was achieved by combining data from both small-angle x-ray and neutron experiments performed on the same samples as well as the incorporation of so-called molecular constraints. Using this model it was found that nanodiscs have an elliptical cross-section, and that the phospholipids in the nanodisc are slightly perturbed, compared to the phospholipids in vesicles. Later, the self-assembly process of nanodiscs was investigated in more detail by varying the self- assembly conditions. Here it was found that the detergent used for solubilizing the phospholipids has a pronounced e↵ect on the self-assembly process. This may be particularly relevant in order to obtain nanodisc samples of high quality and yield, needed for structural studies using small angle scattering. And which has been a challenge during the whole project. In the later part of the project the main work has been on obtaining high quality samples as well as conducting SAXS and SANS measurements on these samples, and finally analyzing the measured data. The data analysis was done using a new hybrid approach combining continuous geometric and discrete modeling schemes. The hybrid approach was used to study the place- ment and orientation of the membrane protein bacteriorhodopsin incorporated into a nanodisc. Furthermore, the hybrid approach also allows for ab. Initio shape reconstruction of membrane proteins of an unknown shape incorporated into a nanodisc. This approach was investigated for the membrane anchored cytochrome P450 3A4 in the final part of the project. iii Resume Phospholipid nanodiscs er 10 nm skiveformede partikler best˚aende af omkring 150 phospho- lipider arrangeret som et dobbeltlag,⇠ stabiliseret af to amfipatiske protein ”bælter” langs kanten af dobbeltlaget. Fordi de indeholder et lille stykke lipid dobbeltlag, hvori et membran protein kan indkorporeres, kan de bruges som et værktøj til studier af membranproteiner. Indtil videre er de fleste studier baseret p˚ananodiscs dog blevet foretaget med henblik p˚afunktionen af det inkorporerede membranprotein. P˚agrund af den gode kontrol af b˚ade størrelsen og lipidsam- mensætningen af nanodisc systemet, burde nanodiscs dog være et ideelt værktøj, til at udvide brugen af sm˚avinkel spredning fra studier af vandopløselige proteiner, til ogs˚aat omfatte mem- branproteiner i nanodiscs. At undersøge mulighederne for dette har været hovedform˚alet for denne afhandling. For at kunne opn˚adette m˚aler det nødvendigt at have en detaljeret forst˚aelse af nanodisc sys- temet uden et inkorporeret membranprotein, samt en forst˚aelse af den selvsanamlings proces hvorved nanodisc fremstilles. Denne forst˚aelse er særligt vigtig i forhold til at optimere inkor- porering af membranproteiner i nanodiscs. At opn˚adenne forst˚aelse var form˚alet i den tidlige fase af projektet. Her blev en detaljeret model for sm˚a-vinkel røntgen og neutron-spredning fra ”tomme” nanodiscs udledt og anvendt til en detaljeret beskrivelse af nanodisc systemet. Den høje grad af detalje blev opn˚aet, ved at kombinere data fra b˚ade røntgen og neutron eksperimenter udført p˚ade samme prøver, samt ved at bruge af s˚akaldte molekylære betingelser i modellen. Ved at anvende denne model blev det observeret, at nanodisc har et elliptisk tværsnit, samt at phospholipiderne i nanodisc’en befinder sig i en let pertuberet tilstand sammenlignet med phospholipid vesikler. Senere i projektet er selvansamlings processen af nanodiscs blev undersøgt nærmere ved at variere de betingelser hvorunder den foreg˚ar. Her blev det konstateret, at den detergent, der anvendes til solubilisering af phospholipiderne, har en kraftig e↵ekt p˚aselvansamling processen. Dette kan især være relevant for at fremstille de nanodisc prøver af høj kvalitet, der er nødvendige for struk- turelle studier ved hjælp af sm˚a-vinkel spredning. Netop at fremstill prøver af en tilstrækkelig høj kvalitet, viste sig at være udfordring igennem hele projektforløbet. I den senere del af projektet har det vigtigste arbejde b˚ade været at opn˚aprøver af en tilstrække- lig høj kvalitet, samt at foretage SAXS og SANS m˚alinger p˚adisse prøver og analysere de m˚alte data. Dataanalysen blev foretaget ved anvende en nyudviklet hybrid metode, der kombinerer en traditionel geometrisk og diskret tilgang. Denne nye hybrid metode benyttes til at undersøge placeringen og orienteringen af membranproteinet bacteriorhodopsin, indkorporeret i en nan- odisc. Desuden giver hybrid metoden ogs˚amulighed for ab. Initio shape reconstruction af mem- branproteiner med en ikke kendt form, indkorporeret i en nanodisc. Dette blev demonstreret for membranproteinet cytokrom P450 3A4, i den sidste del af projektet. iv Preface The work presented in this thesis was done from 2009 to 2012, at the Structural Biophysics (former Biophysics located at the Faculty of Life Sciences) group at the Niels Bohr institute, University of Copenhagen. The main goal has been to develop the phospholipid nanodisc system into a tool for performing structural studies of membrane proteins using small-angle scattering. This has required a very multidisciplinary approach as I have had to learn a number of di↵erent techniques and subjects in addition to the main technique, which has been small-angle scattering. I have performed wet-lab work in order to prepare the nanodisc samples, and participated in several measurements at large scale facilities, such as the ESRF and ILL in France or the PSI in Switzerland. I have written computer programs for analyzing the data and during a one month stay at the university of Illinois, I worked with expressing and purifying the membrane protein Tissue Factor. Although this has been quite a challenge, it has been even more fun. This thesis consists of two parts. The first part introduce the nanodisc system and describe the theoretical background for small angle scattering, setting the stage for the main scientific work presented in the second part. This part consist of 5 papers, of which 3 have been published and 2 are drafts for publications. Included in this part is also a short experimental report of the work done on the membrane protein Tissue Factor. Acknowledgments There are several people that deserve a special thanks for making this an enjoyable experience both on a personal level as well as on an academic level. First of all, I would like to thank my supervisor Lise Arleth for giving me the opportunity to continue the work we started in my master thesis in a Ph. D project, and for always being encouraging and interested. I would like to thank the people that I at some point have had to share my office with: Søren Kynde, Martin Cramer Pedersen, Pie Huda, Jesper Nyaard and Jens B Simonsen: It has been a pleasure. I would like to thank former and present members of the biophysics group: Kell Mortensen, Lars Øendal, Steen L. Hansen, Marianne Jensen, Jacob JK Kirkensgaard, Søren S. Nielsen, Grethe Vestergaard, Rasmus H. Nielsen and Selma Maric. I would also like to thank the people in the Sligar lab at university of Illinois, in particular, Steven Sligar, Illia Denisov and Mark McLean, for letting me loose in the lab. In particular I would like to thank Søren R. Midtgaard for numerous beamtimes and road trips across Europe from one large scale facility to the other. Finally I would like to acknowledge the UNIK - Center For Synthetic Biology, the Graduate School for Molecular Biophysics and the Faculty of Life Sciences for funding of the Ph D project. v Contents Contents 1 Introduction 1 1.1 Motivation ....................................... 1 1.2 Structural Studies of Membrane proteins in Solution . 4 1.3 Results.......................................... 5 2 Nanodiscs 9 2.1 Self-Assembly of Amphiphiles . 9 2.1.1 The Hydrophobic E↵ect . 10 2.1.2 The Critical Micelle Concentration . 11 2.1.3 The Packing Parameter . 12 2.1.4 Mixtures of Amphiphiles . 14 2.1.5 Bilayer Detergent Interactions . 15 2.2 Interactions Between Membrane Proteins and Membranes . 15 2.3 Nanodiscs and Apo-A1 HDL particles . 16 2.3.1 Apo-A1 HDL Particles . 17 2.3.2 Nanodiscs . 20 2.3.3 Self-Assembly of Nanodiscs . 20 2.3.4 Structural Studies of Nanodiscs . 22 2.4 Reconstitution of Membrane Proteins into Nanodiscs . 22 2.4.1 Choice of Detergent for reconstitution of Membrane Proteins .
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