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Triple-Detection Size-Exclusion Chromatography of Membrane Proteins

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Triple-Detection Size-Exclusion Chromatography of Membrane Proteins Triple-Detection Size-Exclusion Chromatography of Membrane Proteins vom Fachbereich Biologie der Technischen Universit¨atKaiserslautern zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Doctor rerum naturalium; Dr. rer. nat.) genehmigte Dissertation von Frau Dipl.-Biophys. Katharina Gimpl Vorsitzende: Prof. Dr. rer. nat. Nicole Frankenberg-Dinkel 1. Gutachter: Prof. Dr. rer. nat. Sandro Keller 2. Gutachter: Prof. Dr. rer. nat. Rolf Diller Wissenschaftliche Aussprache: 08. April 2016 D 386 Abstract Membrane proteins are generally soluble only in the presence of detergent micelles or other membrane-mimetic systems, which renders the determination of the protein's mo- lar mass or oligomeric state difficult. Moreover, the amount of bound detergent varies drastically among different proteins and detergents. However, the type of detergent and its concentration have a great influence on the protein's structure, stability, and func- tionality and the success of structural and functional investigations and crystallographic trials. Size-exclusion chromatography, which is commonly used to determine the molar mass of water-soluble proteins, is not suitable for detergent-solubilised proteins because the protein{detergent complex has a different conformation and, thus, commonly exhibits a different migration behaviour than globular standard proteins. Thus, calibration curves obtained with standard proteins are not useful for membrane-protein analysis. However, the combination of size-exclusion chromatography with ultraviolet absorbance, static light scattering, and refractive index detection provides a tool to determine the molar mass of protein{detergent complexes in an absolute manner and allows for distinguishing the con- tributions of detergent and protein to the complex. The goal of this thesis was to refine the standard triple-detection size-exclusion chro- matography measurement and data analysis procedure for challenging membrane-protein samples, non-standard detergents, and difficult solvents such as concentrated denaturant solutions that were thought to elude routine approaches. To this end, the influence of urea on the performance of the method beyond direct influences on detergents and proteins was investigated with the help of the water-soluble bovine serum albumin. On the basis of the obtained results, measurement and data analysis procedures were refined for different detergents and protein{detergent complexes comprising the membrane proteins OmpLA and Mistic from Escherichia coli and Bacillus subtilis, respectively. The investigations on mass and shape of different detergent micelles and the composi- tions of protein{detergent complexes in aqueous buffer and concentrated urea solutions showed that triple-detection size-exclusion chromatography provides valuable information about micelle masses and shapes under various conditions. Moreover, it is perfectly suited for the straightforward analysis of detergent-suspended proteins in terms of composition and oligomeric state not only under native but, more importantly, also under denaturing conditions. 3 Zusammenfassung Im Allgemeinen sind Membranproteine nur in Detergensmizellen oder anderen membran- mimetischen Systemen l¨oslich, was die Bestimmung von molaren Massen oder Oligomeri- sierungsgraden erschwert. Dar¨uber hinaus variiert die Menge an Detergens, die an ein Protein gebunden ist, stark zwischen verschiedenen Detergentien und Proteinen. Protein- struktur, -stabilit¨at,und -funktion und ebenso der Erfolg von Struktur- und Faltungsstu- dien sowie der Kristallisation von Membranproteinen sind jedoch stark von der Art des De- tergens und seiner Konzentration abhh¨angig.Gr¨oßenausschlusschromatographie (GPC), welche standardm¨aßig f¨urdie Bestimmung von molaren Massen wasserl¨oslicher Proteine Anwendung findet, ist f¨urin Detergens gel¨osteProteine nicht geeignet. Proteindeter- genskomplexe weisen eine andere Konformation auf als globul¨areStandardproteine und unterscheiden sich deshalb f¨urgew¨ohnlich auch in ihrem Migrationsverhalten von diesen. Deshalb k¨onnenmit Standardproteinen erstellte Kalibrierkurven im Allgemeinen nicht f¨ur die Analyse von Membranproteinen verwendet werden. Kombiniert man jedoch GPC mit der Detektion von UV-Extinktion, statischer Lichtstreuung und Brechungsindex, erh¨alt man eine Methode, welche die Bestimmung molarer Massen von Proteindetergenskom- plexen erm¨oglicht und es erlaubt, die Einzelbeitr¨agevon Protein und Detergens zu be- stimmen. Das Ziel dieser Arbeit war es, das Standardmessverfahren und die Datenanalyse dieser Methode f¨urdie Anwendung mit schwierigen Membranproteinproben, außergew¨ohnlichen Detergentien und anspruchsvollen L¨osungsmitteln,wie beispielsweise hochkonzentrierte Denaturierungsmittell¨osungen,zu verfeinern. Dazu wurde der Einfluss von Harnstoff, der von der speziellen Probe unabh¨angigist, auf das Verhalten der Methode an dem wasserl¨oslichen Rinderserumalbumin untersucht. Auf Grundlage der dadurch gewonnenen Erkenntnisse wurden Mess- und Analyseverfahren f¨urverschiedene Detergentien sowie Proteindetergenskomplexe mit den Proteinen OmpLA und Mistic aus Escherichia coli beziehungsweise Bacillus subtilis verfeinert. Die Untersuchungen bez¨uglich der Masse und Form verschiedener Detergentsmizellen sowie der Zusammensetzung der Proteindetergenskomplexe in verschiedenen L¨osungsmit- teln zeigt, dass GPC mit dreifacher Detektion n¨utzliche Informationen ¨uber Masse und Form von Detergensmizellen unter verschiedenen Bedingungen liefert. Dar¨uber hinaus ist die Methode hervorragend geeignet, um detergensgel¨osteProteine bez¨uglich ihres Oligomerisierungsgrades und der Menge des gebundenen Detergens zu charakterisieren, und zwar sowohl unter nativen, als auch unter denaturierenden Bedingungen. 5 Contents Abbreviations and symbols 11 1 Introduction 15 1.1 Membrane proteins . 16 1.2 Model protein: OmpLA . 17 1.3 Model protein: Mistic . 18 1.4 Membrane-mimetic system: detergent micelles . 19 1.4.1 Critical micellar concentration and aggregation number of detergent micelles . 21 1.5 Methods for studying molar masses . 22 1.6 Principles of triple-detection size-exclusion chromatography . 23 1.6.1 Methods for size-based particle separation . 23 1.6.2 Triple-detection . 24 1.7 Goals . 26 2 Theory 29 2.1 Static light scattering . 29 2.2 Multi-component systems . 33 2.3 Mass and size distribution . 35 2.3.1 Molar-mass averages . 35 2.3.2 Mean square radius . 37 2.4 Conformation analysis . 37 3 Materials 39 3.1 Chemicals and enzymes . 39 3.2 Proteins . 39 3.2.1 Mistic . 39 3.2.2 OmpLA . 40 3.3 Detergents . 40 4 Methods 43 4.1 Preparation of denaturants . 43 4.2 Preparation of detergents . 43 4.3 Production and purification of Mistic . 43 4.4 Production and purification of OmpLA . 44 7 8 Contents 4.5 Determination of protein concentrations . 45 4.6 Detergent exchange . 45 4.7 Thin-layer chromatography . 46 4.8 Determination of refractive index increments . 46 4.9 Triple-detection size-exclusion chromatography . 46 4.9.1 Detergents . 47 4.9.2 Bovine serum albumin . 47 4.9.3 Mistic . 48 4.9.4 OmpLA . 48 5 Results 49 5.1 Refractive index increments . 49 5.2 Molar mass determination by triple-detection SEC . 51 5.2.1 Molar mass determination of water-soluble BSA . 51 5.2.2 Sample-to-sample reproducibility . 52 5.3 Behaviour of detergents in triple-detection SEC . 53 5.3.1 Investigation of DDM micelles . 53 5.3.2 Concentration series of alkyl maltosides . 55 5.4 Triple-detection SEC in the presence of urea . 59 5.4.1 BSA in the presence of urea . 59 5.4.2 Alkyl maltoside micelles in the presence of urea . 61 5.5 Separation and characterisation of PDCs and protein-free micelles . 64 5.5.1 BSA in the presence of DDM micelles . 65 5.5.2 LDAO-solubilised OmpLA . 65 5.5.3 Detergent-solubilised Mistic . 66 5.5.4 Detergent-solubilised Mistic in 6 M urea . 68 5.6 Fluorinated octyl maltoside . 71 5.6.1 Mass and shape determination . 71 5.6.2 F6OM mass and shape: theoretical considerations . 74 6 Discussion 77 6.1 Influence of urea on triple-detection SEC . 77 6.1.1 Changes in elution behaviour . 77 6.1.2 Effect on data reproducibility . 79 6.2 Investigation of detergent micelles . 80 6.2.1 Triple-detection SEC as method for micelle size determination . 81 6.2.2 Detector response to detergent micelles . 82 6.2.3 Concentration dependence of elution profiles . 82 6.2.4 Urea-induced changes in micelle size . 83 6.2.5 Determination of micelle shape . 84 Contents 9 6.3 Investigation of PDCs . 86 6.3.1 Composition of PDCs in the presence of urea . 87 6.4 Accuracy of triple-detection SEC . 87 6.5 Limitations of triple-detection SEC . 88 6.6 Outlook . 89 7 Conclusions 91 Bibliography 93 Acknowledgements 109 List of publications 111 Curriculum vitae 113 Eidesstattliche Erkl¨arung 115 Abbreviations and symbols A280 nm absorbance coefficient at 280 nm A2 second virial coefficient abbr. abbreviation AF4 asymmetric-flow field-flow fractionation agg. no. aggregation number AIX anion exchange AUC analytical ultracentrifugation BSA bovine serum albumin BWM bio wet mass C12EO8 octa(ethylene oxide) dodecyl ether CD circular dichroism CF3 centrifugal field-flow fractionation comp protein{detergent complex (subscript) CV column volume DDM n-dodecyl-β-D-maltoside DEAE diethylaminoethanol det detergent (subscript) DIBMA poly(di-isobutylene-alt-maleic-acid) DLPC 1,2-didodecanoyl-sn-glycero-3-phosphocholine DLS dynamic light scattering DM n-decyl-β-D-maltoside DPC n-dodecyl-phosphocholine DTT 1,4-dithio-DL-threitol EDTA ethylenediamine tetraacetic acid EK enterokinase EtOH ethanol
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