Most membrane proteins are inserted into the membrane co-translationally utilizing the translocon, which allows a sufficiently long and hydrophobic stretch of amino acids to partition into the membrane. However, X-ray structures of membrane proteins have revealed that some transmembrane helices (TMHs) are surprisingly hydrophilic. These marginally hydrophobic transmembrane helices (mTMH) are not recognized as TMHs by the translocon in the absence of local sequence context. We have studied three native mTMHs, which were previously shown to depend on a subsequent TMH for membrane insertion. Their recognition was not due to specific interactions. Instead, the presence of basic amino acids in their cytoplasmic loop allowed membrane insertion of one of them. In the other two, basic residues are not sufficient unless followed by another, hydrophobic TMH. Post-insertional repositioning are another way to bring hydrophilic residues into the membrane. We show how four long TMHs with hydrophilic residues seen in X-ray structures, are initially inserted as much shorter membrane-embedded segments. Tilting is thus induced after membrane-insertion, probably through tertiary packing interactions within the protein. Aquaporin 1 illustrates how a mTMH can shape membrane protein folding and how repositioning can be important in post-insertional folding. It initially adopts a four-helical intermediate, where mTMH2 and TMH4 are not inserted into the membrane. Consequently, TMH3 is inserted in an inverted orientation. The final conformation with six TMHs is formed by TMH2 and 4 entering the membrane and TMH3 rotating 180°. Based on experimental and computational results, we propose a mechanism for the initial step in the folding of AQP1: A shift of TMH3 out from membrane core allows the preceding regions to enter the membrane, which provides flexibility for TMH3 to re-insert in its correct orientation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Membrane proteins roughly constitute 30% of open reading frames in a genome and form 70% of current drug targets. They are classified as integral, peripheral membrane proteins and polypeptide toxins. α-helices and β -strands are the principal secondary structures observed in integral membrane proteins. This thesis presents the results of studies on analysis and correlation of sequence and structure of helices constituting integral helical membrane proteins. The aim of this work is to understand the helix stabilization, distortion as well as packing in terms of amino acid sequences and the correlated structures they adopt. To this end, analyses of datasets of X-ray crystal structures of integral helical membrane proteins and their comparison with a dataset of representative folds of globular proteins was carried out. Initial analysis was carried out using a non-redundant dataset of 75 membrane proteins to understand sequence and structural preferences for stabilization of helix termini. The subsequent analysis of helix distortions in membrane proteins was carried out using an updated dataset of 90 membrane proteins. Chapter 1 of the thesis reviews experimental as well as theoretical studies that have provided insights into understanding the structure of helical membrane proteins. Chapter 2 details the methods used during the course of the present investigations. These include the protocol used for creation of the non-redundant database of membrane and globular proteins. Various statistical methods used to test significance of the position-wise representation of amino acids in helical regions and the differences in globular and membrane protein datasets have been listed. Based on the tests of significance, a methodology to identify differences in propensity values that are statistically significant among two datasets has been devised. Programs used for secondary structure identification of membrane proteins namely Structure Identification (STRIDE) and Assignment of Secondary Structure in Proteins (ASSP) as well as those used for characterization of helical geometry (Helanal-Plus) have also been enlisted. In Chapter 3, datasets of 865 α-helices in 75 membrane proteins and 2680 α- helices from 626 representative folds in globular proteins defined by the STRIDE program have been analyzed to study the sequence determinants at fifteen positions within and around the α-helix. The amino acid propensities have been studied for positions that are important for the process of helix initiation, propagation, stabilization and termination. Each of the 15 positions has unique sequence characteristics reflecting their role and contribution towards the stability of the α-helix. A comparison of the sequence preferences in membrane and globular proteins revealed common residue preferences in both these datasets confirming the importance of these positions and the strict residue preferences therein. However, short/medium length α-helices that initiated/terminated within the membrane showed distinct amino acid preferences at the N-terminus (Ncap, N1, N2) as well as the C-terminus ( Ccap, Ct) when compared to α-helices belonging to membrane and globular proteins. The sequence preferences in membrane proteins were governed by the helix initiating and terminating property of the amino acids as well as the external environment of the helix. Results from our analysis also conformed well with experimentally tested amino acid preferences in a position-specific amino acid preference library of the rat neurotensin receptor (Schlinkmann et al (2012) Proc Natl Acad Sci USA 109(25):1890-5) as well as crystal structures of GPCR proteins. In the light of the environment dependent amino acid preferences found at α- helix termini, a survey was carried out to find various helix capping motifs adopted at both termini of α-helices in globular and membrane proteins to stabilize these helix termini. The results from these findings have been reported in Chapter 4. A sequence dependent structural preference is found for capping motifs at helix termini embedded inside and protruding outside the membrane. The N-terminus of α-helices was capped by hydrogen bonds involving free main chain amide groups of the first helical turn as donors and amino acid side chains as acceptors, as against the C-terminus which showed position-dependent characteristic backbone conformations to cap the helix. Overall helix termini inside the membrane did not show a very high number of capping motifs; instead these termini were stabilized by helix- helix interactions contributed by the neighboring helices of the helical bundle. In Chapter 5, we examine transmembrane helical (TMH) regions to identify as well as characterize the various types of helix perturbations in membrane proteins using ASSP and Helanal-Plus. A survey of literature shows that the term ‘helix kink’ has been used rather loosely when in fact helical regions show significant amounts of variation and transitions in helical parameters. Hence a systematic analysis of TMH regions was undertaken to quantify different types of helix perturbations, based on geometric parameters such as helical twist, rise per residue and local bending angle. Results from this analysis indicated that helices are not only kinked but undergo transitions to form interspersed stretches of 310 helices and π-bulges within the bilayer. These interspersed 310 and π-helices showed unique sequence preferences within and around their helical body, and also assisted in main- taining the helical structure within the bilayer. We found that Proline not only kinked the helical regions in a characteristic manner but also caused a tightening or unwinding in a helical region to form 310 and π-helix fragments respectively. The helix distortions also resulted in backbone hydrogen bonds to be missed which were stabilized by hydrogen bonds from neighboring residues mediated by their side chain atoms. Furthermore, a packing analysis showed that helical regions with distortions were able to establish inter-helical interactions with more number of transmembrane segments in the helical bundle. The study on helix perturbations presented in the previous chapter, brought to light a previously unreported 19 amino acid π-helix fragment interspersed between α-helices in the functionally important transmembrane helix 2 (TM2) belonging to Mitochondrial cytochrome-c-oxidase (1v55). Chapter 6 describes a case study of the structurally similar but functionally different members within the Heme-Copper- Superoxidases (HCO) superfamily that were considered for a comparative analysis of TM2. An analysis of 7 family members revealed that the π-helix shortens, fragments in two shorter π-helices or was even absent in some family members. The long π-helix significantly decreased the total twist and rise of the entire helical fragment thus accommodating more hydrophobic amino acids within the bilayer to avoid hydrophobic mismatch with the bilayer. The increased radius of the TM2 helical fragment also assisted in helix packing interactions by increasing the number of residues involved in helix-helix interactions and hydrogen bonds. Chapter 7 documents the conclusions from the different analyses presented in each of the above chapters. Overall, it is found that membrane proteins optimize the biophysical and chemical constraints of the external environment to strategically place select amino acids at helix termini to ‘start’ and ‘stop’ α-helices. The stabilization of these helix termini is a consequence of sequence dependent structural preferences to form helix capping motifs. The studies on helix transitions and distortions highlight that membrane proteins are not only packed as α-helices but also accomodate 310- and π-helical fragments. These transitions and distortions help in harboring more hydrophobic amino acids and aiding inter-helical interactions important for maintaining the fold of the membrane protein. Appendix A describes a comparison of α-helix assignments in globular and membrane proteins by two algorithms, one based on Cα trace (ASSP) and the other using a combination of hydrogen bond pattern along with backbone torsion angles φ and ψ (STRIDE).

Membrane proteins are of undeniable importance for cell physiology across all domains of life and a loss of their function, e.g., due to mutations in their coding sequence, is almost always linked to disease. In humans, mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-gated anion channel in epithelia, give rise to cystic fibrosis (CF). Over 2100 mutations of the CFTR gene are known, however, their disease liability remains mostly undetermined. Causal therapies, i.e., small-molecule drugs that target CFTR itself, have improved the lives of people with the most common mutations (e.g. ΔF508, G551D) over the last decade. In contrast, many rare CF-phenotypic mutations are not eligible for these novel treatments and would benefit from in vitro evaluation of their molecular consequences. In vitro studies of membrane proteins are often complicated by the intrinsic hydrophobicity and aggregation susceptibility of this protein group. However, this can be avoided by using short membrane protein fragments corresponding to the smallest in vivo folding unit of the respective protein at the ER membrane. These model proteins can be easily genetically modified, expressed and purified, making them a suitable tool to pinpoint the effects of mutations. This thesis demonstrates the utility of such a reductionist model system: TM3/4, the second helical hairpin of CFTR’s transmembrane domain 1, was used to study protein folding with a focus on disease-causing missense mutations of CFTR, which may cause CFTR misfolding in vivo. TM3/4 purification was first optimized by using a thioredoxin tag, which allowed heat purification of the fusion protein even after initial purification steps. Optimal heat treatment for maximal protein purity and recovery were determined for TM3/4 and another helical hairpin, ATP synthase subunit c. Moreover, tertiary folding of a CF-phenotypic loop mutation, E217G, introducing a non-native GXXXG interaction motif was analyzed by single-molecule Förster resonance energy transfer (smFRET) in different lipid bilayer conditions, showing unusually increased stability in comparison to wild type (WT) TM3/4. Furthermore, smFRET was used in tandem with circular dichroism and fluorescence spectroscopy to assess the effect of a specific membrane lipid, cholesterol, on TM3/4 variants showing significant changes on secondary but not tertiary structure. Lastly, a mutant library of 13 TM3/4 mutants was established to perform drug screenings with CFTR correctors – a class of small molecules rescuing or preventing misfolding of CFTR. This screening study demonstrated that (i) not all CF-phenotypic missense mutations are locally misfolded at a lipid bilayer comparable to the ER membrane; and (ii) in vitro restoration of a native WT-like conformation of locally misfolded TM3/4 mutants is not only possible but different drug-mutant pairings can be identified related to folding rescue efficiency of a given corrector on a respective mutant. The latter identified drug-mutant pairings may lead to drug repurposing if the effect can be confirmed in cell culture experiments. In conclusion, the TM3/4 minimal model of CFTR and biophysical methods, such as smFRET, proved as versatile tools not only for investigation of mutation and lipid effects on membrane protein folding but also for drug screenings in a disease context.:1 INTRODUCTION 2 THEORETICAL BACKGROUND 2.1 MEMBRANE PROTEINS AND THEIR NATIVE ENVIRONMENTS 2.1.1 Membrane protein families and their role in human health 2.1.2 Fundamental folding models of α-helical membrane proteins 2.1.3 Co-translational folding at the ER supported by the translocon 2.1.4 Folding-relevant interactions within membrane proteins 2.1.5 Biological membranes and lipid classes 2.1.6 Physical properties of lipid bilayers impacting membrane proteins 2.1.7 Membrane models for in vitro studies 2.2 CYSTIC FIBROSIS AND CFTR 2.2.1 Pathology of cystic fibrosis 2.2.2 Structure and function of the CFTR channel 2.2.3 A minimal model of CFTR to study rare CF mutations 2.2.4 Missense mutations within the CFTR segmental model TM3/4 2.2.5 Novel modulator therapies for the treatment of cystic fibrosis 2.3 IN VITRO ASSESSMENT OF MEMBRANE PROTEIN FOLDING 2.3.1 Expression and purification of membrane proteins 2.3.2 Single-molecule FRET in single- and multi-well mode for protein folding 3 HEAT PURIFICATION OF TRX MEMBRANE PROTEIN FUSIONS 3.1 PREAMBLE AND SUMMARY 3.2 RESULTS AND DISCUSSION 4 IMPACT OF A CFTR LOOP MUTATION WITH ATYPICAL STABILITY 4.1 PREAMBLE AND SUMMARY 4.2 RESULTS AND DISCUSSION 5 EFFECTS OF CHOLESTEROL ON LOCAL CFTR FOLDING 5.1 PREAMBLE AND SUMMARY 5.2 RESULTS 5.2.1 Folding of TM3/4 hairpins in the presence of cholesterol 5.2.2 Folding of TM3/4 hairpins in the presence of Lumacaftor 5.2.3 Impact of Lumacaftor on membrane fluidity 5.3 DISCUSSION 6 CFTR CORRECTOR SCREENINGS WITH SINGLE-MOLECULE FRET 6.1 PRESCREENING TO IDENTIFY MISFOLDED TM3/4 VARIANTS 6.2 SCREENING OF MISFOLDED TM3/4 VARIANTS WITH CFTR CORRECTORS 7 CONCLUSIONS 8 OUTLOOK 9 MATERIALS AND METHODS 9.1 CONSTRUCT DESIGN OF HELICAL TRANSMEMBRANE HAIRPINS 9.2 PROTEIN EXPRESSION AND PURIFICATION 9.3 HEAT TREATMENT OF HELICAL TRANSMEMBRANE CONSTRUCTS 9.4 SINGLE-MOLECULE FRET EXPERIMENTS 9.4.1 Labeling of TM3/4 constructs 9.4.2 Liposome preparation and reconstitution of labeled protein constructs 9.4.3 Single-molecule FRET measurements in manual mode 9.4.4 Single-molecule FRET measurements in multi-well screening mode 9.5 CIRCULAR DICHROISM SPECTROSCOPY 9.5.1 Circular dichroism to determine protein heat stability 9.5.2 Circular dichroism to study protein structure in different lipid bilayers 9.6 FLUORESCENCE SPECTROSCOPY 9.6.1 Vesicle leakage assay to test lipid bilayer stability 9.6.2 Examining lipid bilayer fluidity with fluorescent probes 10 APPENDIX 10.1 GENERATION OF A TM3/4 MUTANT LIBRARY 10.2 TM3/4 SCREENINGS WITH CFTR CORRECTORS 10.2.1 SmFRET control screenings and supporting data 10.2.2 Extracted closed state fractions from smFRET screenings 10.2.3 DLS to measure vesicle integrity after corrector addition 11 REFERENCES 12 ACKNOWLEDGEMENTS 13 ERKLÄRUNG GEMÄß §5 ABS. 1 S. 3 DER PROMOTIONSORDNUNG / Membranproteine sind für die Zellphysiologie aller biologischen Domänen von unbestreitbarer Bedeutung und ein Verlust ihrer Funktion, z.B. durch Mutationen in ihrer kodierenden Sequenz, ist fast immer Auslöser von Krankheiten. Beim Menschen führen Mutationen im Gen für den Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), einen ATP-abhängigen Anionenkanal in Epithelien, zu Mukoviszidose (CF). Über 2100 Mutationen des CFTR-Gens sind bekannt – ob jedoch alle Mutationen tatsächlich CF auslösen, ist weitgehend ungeklärt. Kausale Therapien, d.h. niedermolekulare Medikamente, die auf CFTR selbst abzielen, haben in den letzten zehn Jahren die Lebensqualität von Menschen mit den häufigsten Mutationen (z.B. ΔF508, G551D) verbessert. Demgegenüber stehen jedoch viele seltene CF-phänotypische Mutationen, für welche diese neuartigen Behandlungen nicht zugelassen sind, wodurch diese Mutationen von einer In-vitro-Analyse ihrer molekularen Konsequenzen profitieren würden. In-vitro-Untersuchungen von Membranproteinen werden oft durch die intrinsische Hydrophobizität und Aggregationsanfälligkeit dieser Proteine erschwert. Dies kann jedoch vermieden werden, indem kurze Membranproteinfragmente verwendet werden, die der kleinsten in vivo Faltungseinheit des jeweiligen Proteins an der ER-Membran entsprechen. Diese Modellproteine können routiniert genetisch verändert, exprimiert und aufgereinigt werden, was sie zu einem geeigneten Werkzeug macht, um die Auswirkungen von Mutationen zu genau festzustellen. Diese Dissertation demonstriert die Nützlichkeit eines solchen reduktionistischen Modellsystems: TM3/4, das zweite helikale Haarnadel-Motiv der Transmembrandomäne 1 von CFTR, wurde verwendet, um Proteinfaltung mit Schwerpunkt auf krankheitsverursachende Missense-Mutationen von CFTR zu untersuchen, welche eine CFTR-Fehlfaltung in vivo verursachen können. Die TM3/4-Aufreinigung wurde zunächst durch die Verwendung eines Thioredoxin-Tags optimiert, der eine Hitzeaufreinigung des Fusionsproteins auch nach anfänglichen Reinigungsschritten ermöglichte. Die optimale Hitzebehandlung für maximale Proteinreinheit und -ausbeute wurde für TM3/4 und ein weiteres helikales Haarnadelprotein, die ATP-Synthase-Untereinheit c, bestimmt. Weiterhin wurde die tertiäre Faltung einer CF-phänotypischen Mutation, E217G, die ein nicht-natives GXXXG-Interaktionsmotiv einführt, mittels einzelmolekularem Förster-Resonanzenergietransfer (smFRET) in verschiedenen Lipiddoppelschichten analysiert, welche eine ungewöhnlich erhöhte Stabilität im Vergleich zum TM3/4-Wildtyp (WT) zeigte. Darüber hinaus wurde smFRET in Verbindung mit Circulardichroismus und Fluoreszenzspektroskopie verwendet, um die Wirkung eines spezifischen Membranlipids, Cholesterin, auf TM3/4-Varianten zu untersuchen, welches signifikante Auswirkungen auf die sekundäre, aber nicht auf die tertiäre Proteinstruktur hatte. Schließlich wurde eine Mutantenbibliothek von 13 TM3/4-Mutanten eingerichtet, um Wirkstoffscreenings mit CFTR-Korrektoren durchzuführen – einer Klasse kleiner Moleküle, die die Fehlfaltung von CFTR verhindern können. Diese Screening-Studie zeigte, dass (i) nicht alle CF-phänotypischen Missense-Mutationen lokal an einer Lipiddoppelschicht fehlgefaltet sind, die mit der ER-Membran vergleichbar ist; und (ii) die In-vitro-Wiederherstellung einer nativen WT-ähnlichen Konformation von lokal fehlgefalteten TM3/4-Mutanten ist nicht nur möglich, sondern es können auch verschiedene Wirkstoff-Mutanten-Paare identifiziert werden, die mit der Faltungsrettungseffizienz eines Korrektors auf eine bestimmte Mutante zusammenhängen. Die letztgenannten Wirkstoff-Mutanten-Paare können zu Drug-Repurposings führen, wenn die Wirkung in Zellkulturexperimenten bestätigt werden kann. Im Allgemeinen, haben sich das TM3/4-Minimalfaltungsmodell von CFTR sowie biophysikalische Methoden, wie z.B. smFRET, als vielseitige Werkzeuge nicht nur für die Untersuchung von Mutations- und Lipideffekten auf die Membranproteinfaltung, sondern auch für das Screening von Medikamenten im Krankheitskontext erwiesen.:1 INTRODUCTION 2 THEORETICAL BACKGROUND 2.1 MEMBRANE PROTEINS AND THEIR NATIVE ENVIRONMENTS 2.1.1 Membrane protein families and their role in human health 2.1.2 Fundamental folding models of α-helical membrane proteins 2.1.3 Co-translational folding at the ER supported by the translocon 2.1.4 Folding-relevant interactions within membrane proteins 2.1.5 Biological membranes and lipid classes 2.1.6 Physical properties of lipid bilayers impacting membrane proteins 2.1.7 Membrane models for in vitro studies 2.2 CYSTIC FIBROSIS AND CFTR 2.2.1 Pathology of cystic fibrosis 2.2.2 Structure and function of the CFTR channel 2.2.3 A minimal model of CFTR to study rare CF mutations 2.2.4 Missense mutations within the CFTR segmental model TM3/4 2.2.5 Novel modulator therapies for the treatment of cystic fibrosis 2.3 IN VITRO ASSESSMENT OF MEMBRANE PROTEIN FOLDING 2.3.1 Expression and purification of membrane proteins 2.3.2 Single-molecule FRET in single- and multi-well mode for protein folding 3 HEAT PURIFICATION OF TRX MEMBRANE PROTEIN FUSIONS 3.1 PREAMBLE AND SUMMARY 3.2 RESULTS AND DISCUSSION 4 IMPACT OF A CFTR LOOP MUTATION WITH ATYPICAL STABILITY 4.1 PREAMBLE AND SUMMARY 4.2 RESULTS AND DISCUSSION 5 EFFECTS OF CHOLESTEROL ON LOCAL CFTR FOLDING 5.1 PREAMBLE AND SUMMARY 5.2 RESULTS 5.2.1 Folding of TM3/4 hairpins in the presence of cholesterol 5.2.2 Folding of TM3/4 hairpins in the presence of Lumacaftor 5.2.3 Impact of Lumacaftor on membrane fluidity 5.3 DISCUSSION 6 CFTR CORRECTOR SCREENINGS WITH SINGLE-MOLECULE FRET 6.1 PRESCREENING TO IDENTIFY MISFOLDED TM3/4 VARIANTS 6.2 SCREENING OF MISFOLDED TM3/4 VARIANTS WITH CFTR CORRECTORS 7 CONCLUSIONS 8 OUTLOOK 9 MATERIALS AND METHODS 9.1 CONSTRUCT DESIGN OF HELICAL TRANSMEMBRANE HAIRPINS 9.2 PROTEIN EXPRESSION AND PURIFICATION 9.3 HEAT TREATMENT OF HELICAL TRANSMEMBRANE CONSTRUCTS 9.4 SINGLE-MOLECULE FRET EXPERIMENTS 9.4.1 Labeling of TM3/4 constructs 9.4.2 Liposome preparation and reconstitution of labeled protein constructs 9.4.3 Single-molecule FRET measurements in manual mode 9.4.4 Single-molecule FRET measurements in multi-well screening mode 9.5 CIRCULAR DICHROISM SPECTROSCOPY 9.5.1 Circular dichroism to determine protein heat stability 9.5.2 Circular dichroism to study protein structure in different lipid bilayers 9.6 FLUORESCENCE SPECTROSCOPY 9.6.1 Vesicle leakage assay to test lipid bilayer stability 9.6.2 Examining lipid bilayer fluidity with fluorescent probes 10 APPENDIX 10.1 GENERATION OF A TM3/4 MUTANT LIBRARY 10.2 TM3/4 SCREENINGS WITH CFTR CORRECTORS 10.2.1 SmFRET control screenings and supporting data 10.2.2 Extracted closed state fractions from smFRET screenings 10.2.3 DLS to measure vesicle integrity after corrector addition 11 REFERENCES 12 ACKNOWLEDGEMENTS 13 ERKLÄRUNG GEMÄß §5 ABS. 1 S. 3 DER PROMOTIONSORDNUNG