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INSERTION STUDIES OF MODEL TRANSMEMBRANE SEGMENTS INTO BACTERIAL AND EUKARYOTIC MEMBRANES
Nina Schiller
Insertion studies of model transmembrane segments into bacterial and eukaryotic membranes
Nina Schiller ©Nina Schiller, Stockholm University 2017
ISBN print 978-91-7797-002-6 ISBN PDF 978-91-7797-003-3
Printed in Sweden by Universitetsservice US-AB, Stockholm 2017 Distributor: Department of Biochemistry and Biophysics, Stockholm University To my family
Contents
List of publications ...... 1
Additional publications ...... 2
Abstract ...... 3
Sammanfattning på svenska ...... 4
Introduction ...... 5
1 The prokaryotic cell ...... 7
2 The eukaryotic cell ...... 9
3 Biological membranes ...... 11 3.1 General features of biological membranes ...... 11 3.2 Different types of biological membranes ...... 12 3.3 The bacterial membrane ...... 14 3.4 The endoplasmic reticulum ...... 15
4 Proteins inside or at the surface of the membrane ...... 17 4.1 ȕ-barrel membrane proteins ...... 19 4.2 Į-helical membrane proteins ...... 19 4.3 Protein-lipid interactions ...... 20
5 Protein biogenesis ...... 22 5.1 Protein synthesis within the ribosome...... 22
6 Protein targeting and transport ...... 24 6.1 Signal sequence ...... 24 6.2 Protein targeting to the bacterial inner membrane and the ER ...... 25 6.2.1 The co-translational protein targeting pathway ...... 26 6.2.2 Post-translational protein targeting pathway ...... 27
7 Protein translocation machineries ...... 29 7.1 The Sec translocon ...... 29 7.2 Accessory components ...... 31 7.2.1 Bacterial SecDFYajC and YidC...... 31 7.2.2 Eukaryotic TRAM, Calnexin, Calreticulin, OST, PDI ...... 31 7.3 Signal peptidase ...... 33 8 ,QWHJUDWLRQRIĮ-helical membrane proteins into the membrane ...... 34
9 3URSHUWLHVRIĮ-helical transmembrane segments ...... 35 9.1 Polar amino acids in transmembrane segments ...... 35 9.2 Proline and glycine in transmembrane segments ...... 36 9.3 The “aromatic belt” ...... 36 9.4 “Snorkeling” ...... 36
10 Membrane protein topology ...... 38 10.1 The positive inside rule ...... 39 10.2 Lipids as topology determinants ...... 40 10.3 Other topology determinants ...... 40 10.4 Hydrophobic mismatch ...... 41 10.5 Re-entrant regions ...... 41 10.6 Topology mapping...... 41 10.6.1 PhoA ...... 41 10.6.2 GFP ...... 42 10.6.3 Glycosylation mapping ...... 42 10.6.4 Protease-protection assay ...... 42 10.6.5 Epitope tags ...... 42 10.7 Topology predictions ...... 43 10.7.1 Hydrophobicity scales ...... 43
11 )ROGLQJRIĮ-helical membrane proteins in the membrane ...... 46 11.1 Helix-helix interactions ...... 47 11.2 van der Waals interactions ...... 47 11.3 Salt bridges ...... 47 11.4 Hydrogen bonds ...... 47 11.5 The GxxxG motif ...... 48 11.6 Knobs-into-holes packing ...... 48
12 Translational arrest peptides ...... 49 12.1 The biology of arrest peptides ...... 50 12.2 The ribosome exit tunnel ...... 51 12.3 Interactions between the ribosomal tunnel and passing nascent chains ..... 52 12.4 Nascent-chain induced translational stalling ...... 52 12.5 Features of arrest peptides ...... 53 12.5.1 The SecM arrest peptide...... 53 12.5.2 The XBP1 arrest peptide...... 54 12.5.3 Release of arrest-peptide mediated translational stalling ...... 55 12.5.4 Other amino acid clusters can arrest translation ...... 55 12.5.5 Laboratory-engineered arrest peptides ...... 56
13 Methodology ...... 57 13.1 Leader peptidase – a model protein (Papers I-IV) ...... 57 13.2 In vitro protein expression (Papers I-IV) ...... 58 13.3 Lep constructs (Papers I-IV) ...... 58 13.4 Determining the propensity of hydrophobic segments to integrate into the ER membrane by in vitro experiments (Papers I, II) ...... 59 13.5 “Pulling force” measurements in vitro and in vivo with the use of arrest peptides (Papers III, IV) ...... 60 13.6 Determination of the minimal glycosylation distance ...... 61 13.7 Circular Dichroism Spectroscopy ...... 61 13.8 Reversed-Phase High-Performance Liquid Chromatography (Papers I, II) . 62
14 Summary of publications ...... 63 14.1 Paper I - Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences ...... 63 14.2 Paper II - Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo ...... 64 14.3 Paper III - A Biphasic Pulling Force Acts on Transmembrane Helices During Translocon-Mediated Membrane Integration ...... 65 14.4 Paper IV – Mutational Analysis of the Human XBP1 Translational Arrest Peptide and Construction of Arrest-Enhanced Variants ...... 65
15 Final thoughts ...... 67
16 Acknowledgements ...... 68
References ...... 70
List of publications
Paper I - Tracy A. Stone, Nina Schiller, Gunnar von Heijne and Charles M. Deber. Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences. Biochemistry. 2015 Feb 24;54(7):1465-73.
Paper II - Tracy A. Stone*, Nina Schiller*, Natalie Workewych, Gunnar von Heijne and Charles M. Deber. Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo. Biochemistry. 2016 Oct 11;55(40):5772-5779.
Paper III - Nurzian Ismail, Rickard Hedman, Nina Schiller and Gunnar von Heijne. A biphasic pulling force acts on transmembrane helices during translocon-mediated membrane integration. Nat Struct Mol Biol. 2012 Oct;19(10):1018-22.
Paper IV – Nina Schiller, Anastasia Magoulopoulou, Florian Cymer, Robert Daniels and Gunnar von Heijne. Mutational analysis of the human XBP1u translational arrest peptide and construction of arrest-enhanced variants. Manuscript.
* These authors contributed equally.
1 Additional publications
Paper V - Martin B. Ulmschneider, Jakob P. Ulmschneider, Nina Schiller, B. A. Wallace, Gunnar von Heijne and Stephen H. White. Spontaneous transmembrane helix insertion thermodynamically mimics translocon-guided insertion. Nat Commun. 2014 Sep 10;5:4863.
Paper VI - Eriko Mishima, Yoko Sato, Kei Nanatani, Naomi Hoshi, Jong- Kook Lee, Nina Schiller, Gunnar von Heijne, Masao Sakaguchi and Nobuyuki Uozumi. The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel. Biochem J. 2016 Dec 1;473(23):4361-4372.
2 Abstract
Cells are encapsulated by a biological membrane in order to separate the cell interior from the surrounding environment. Different lipids and proteins compose the membrane and present a semi-permeable barrier for the diffusion of ions and molecules across the lipid bilayer. Membrane proteins also mediate the passage of signals between the interior and the exterior of the cell. To ensure the proper functioning of membrane proteins, it is essential that nascent membrane proteins are correctly integrated into the lipid bilayer to be able to fold and oligomerize. In this thesis, an engineered protein containing two natural transmembrane segments followed by an additional test segment, has been used as a model protein to study (i) sequence requirements for translocon-mediated insertion of the test segment, (ii) dynamics of nascent membrane proteins undergoing translocon-mediated insertion and (iii) to carry out an extensive mutagenesis scan to identify critical residues in the mammalian arrest peptide Xbp1 that enhances translational stalling in the ribosome. This provides a toolbox of arrest peptides with different stalling strengths that will be useful for force measurements on nascent protein chains.
3 Sammanfattning på svenska
Alla levande organismer innehåller proteiner. Proteiner är kedjemolekyler uppbyggda av aminosyror med olika kemiska och fysikaliska egenskaper, som i sin tur ger olika proteiner olika karaktärer. Proteinernas funktioner kan vara allt ifrån att kopiera cellens arvsmassa inför nya cellgenerationer till att transportera avfallsprodukter eller näringsämnen in och ut genom cellens yttersta barriär, cellmembranet. I celler finns det ”proteinfabriker” – ribosomer – som tillverkar alla cellens proteiner. Medan proteinsyntesen fortgår kan yttre faktorer i den omgivande miljön utanför ribosomens proteinskyddande väggar påverka den nysyntetiserade proteindelen. Till exempel kan den färdigsyntetiserade proteindelen börja interagera med andra komponenter i cellen, som i sin tur kan guida det ribosombundna proteinet till olika delar av cellen. Proteiner som sitter i cellmembranet har en vattenavvisande hydrofob karaktär. Membranproteiner sätts normalt in i membranet medan proteinsyntesen fortfarande pågår, för att minimera tiden proteinet befinner sig i cellvätskan, cytoplasman. I denna avhandling har vi undersökt dels hur kompositionen av aminosyror i membranproteiner kan påverka membraninsättningsförmågan, dels vilka krafter som verkar på ett membranprotein under dess färd in i cellmembranet. Vi har även skapat ett bibliotek av varianter av korta aminosyrekedjor, s.k. arrestpeptider, som besitter egenskapen att pausa proteinsyntesen i ribosomen. Våra resultat visar att peptidsegment som innehåller hydrofoba aminosyror i en följd efter varandra har en högre förmåga att integreras i cellmembranet, jämfört med segment där de hydrofoba aminosyrorna är utspridda (Artikel I). Vidare att denna effekt bara ses för peptidsegment som är ”marginellt hydrofoba”, dvs. varken för hydrofoba eller för hydrofila (Artikel II). Vi har också funnit att starka krafter påverkar membranproteiner under deras insättningsprocess in i cellmembranet, både i prokaryota och eukaryota celler (Artikel III). Krafterna uppmättes med hjälp av av prokaryota och eukaryota arrestpeptider. För att bredda användningsområdet för eukaryota arrestpeptider har vi skapat ett arrestpeptidbibliotek innehållande en stor variation av arrestpeptider av varierande styrka. Dessa kan användas för framtida studier av t.ex. protein-protein-interaktioner eller proteinveckning.
4 Introduction
All living organisms consist of structural and functional units called cells. Scientists try to understand these basic, but ever so complex, units of living matter. Arguably, we have better knowledge about the organization of the universe than the processes of living cells. We can calculate the Sun’s age and know when it will end to shine, but we cannot explain why a human being can live for eighty years but a mouse for only two. Thanks to advances in biotechnology we have obtained whole genome sequences for these and many other species, but still we cannot second-guess how a cell behaves when we manipulate a previously unstudied gene. Stars are 1043 times bigger than cells, but these individual and independent unicellular organisms have a more intricate structure that builds up more astonishing complex machines through the natural processes within the laws of physics and chemistry. During about 20 % of the age of the universe, cells have progressively undergone evolutionary refinement, leading to diverse specializations of remarkable “molecular chemical factories”. 3.8 billion years ago the formation of some kind of lipid membranes in an aqueous environment set the ground for what we know as life today. The evolutionary process of compartmentalization permitted the cell to maintain a barrier separating the interior part of the cell from the exterior surroundings, while at the same time allowing matter, information and energy to flow across the membrane through specialized membrane- embedded proteins. These integral membrane proteins are the focus in this thesis. In most proteomes, 20-30 % of the proteins are such membrane proteins; importantly, membrane proteins are the targets for approximately 60 % of pharmaceutical drugs. In order for membrane proteins to perform their functions, they need to arrive at their designated membrane, where they can acquire an active functional structure and further interact with other membrane proteins. In order to understand the full life cycle of a membrane protein it is important to investigate all the steps they pass through, ranging from its birth, when it is synthesized by the ribosome and integrated into a membrane, until its death through degradation by proteases. This thesis includes four papers that study the “birth” of membrane proteins, in particular how they integrate into both bacterial and eukaryotic cell membranes. A general background to biological membranes, membrane proteins, and membrane protein
5 biosynthesis is followed by a brief description of the methodology used in the thesis, and a summary of the main results of the thesis work.
6
2 The eukaryotic cell
Eukaryotes range in from small sized single-cell organisms such as the yeast Saccharomyces cerevisiae, to large multi-cellular organisms such as Homo sapiens, which consists of 1013 cells. The eukaryotic cell contains a large number of different organelles, membrane-enclosed sub-compartments that are specialized in various aspects of the cell metabolism. All eukaryotic cells store their DNA in the nucleus, translocate and transport proteins to their final cellular locations through the ER and Golgi organelles and produce energy carrier molecules for the cell in the form of ATP in the mitochondria. Additional organelles are vacuoles and lysosomes that carry out degradation processes of macromolecules, and peroxisomes with the major role of long chain fatty acid breakdown and destruction of hydrogen peroxide. The eukaryotic plant cell also houses light-harvesting and ATP-producing chloroplasts (Fig 3). The various organelles with their distinguishable lipid bilayers contain their specific complement of proteins. In order for the proteins to target correctly to their destined membrane or subcellular organelle, amino acid sequences have evolutionary been developed to guide the protein in their cellular journey (6-8).
9
membrane components and can play an essential role for protein structure and function (12-15). Intracellular organelles have a distinct and functional architecture with regions of highly curved and bent bilayers. The major players contributing to introducing these high membrane curvatures appear to be membrane proteins. The asymmetric lipid distribution in the bilayer can also generate curvature, but to a lesser degree (16). There are two mechanisms for inducing local membrane bending. The first one is named wedging, which describes the disturbed lipid packing in the two bilayer leaflets as a hydrophobic domain of a protein gets inserted into only one leaflet. The second is named scaffolding, where scaffolding proteins adopt an inflexible curved structure and bind tightly to a membrane surface, forcing the membrane into the inherent structure of the scaffold (17).
3.2 Different types of biological membranes
The cell membrane contains about 5 × 106 lipids molecules in a 1 μm × 1 μm area (1). Different types of lipid composition contribute to a variety of physical properties of membranes. Membrane thickness, surface charge, fluidity and curvature are global properties that can differ. Environmental changes also influence these characteristics, requiring a diversity of lipids to allow the cell to adapt to environmental changes (18-20). Despite the chemical diversity, most lipids share common structural features, such as one polar and one non-polar part. The most abundant class of lipids in cell membranes are phospholipids, with the main one being phosphoglycerides. They contain a polar head group and a hydrophobic tail consisting of two fatty acyl chains. The long acyl chains are attached to through ester bonds to glycerol (18) (Fig. 5).
12
% phosphatidylethanolamine (PE), 20-25 % phosphatidylglycerol (PG) and 5-10 % cardiolipin (28, 29).
3.4 The endoplasmic reticulum
The ER is an organelle in eukaryotic cells organized in a network of membrane-enclosed tubules and cisternae. The membrane separates the interior (or lumen) from the cytosol. The ER serves as a platform for many cellular processes, involving protein synthesis, folding, protein quality control and lipid synthesis. The ER network is in constant dynamic exchange with the nuclear membrane, mitochondrial membranes, the Golgi membrane and peroxisomal membranes (30). These contacts are important to enable direct transport of lipids from the ER to mitochondria and peroxisomes (31, 32). Besides the many cellular functions the ER is involved in, it additionally has important roles in Ca2+ signaling, drug metabolism, and membrane protein biosynthesis (33). In the ER the lipid distribution is similar in both leaflets. However, in the plasma membrane the lipid distribution is asymmetric between the two leaflets (20). It has been suggested that the maintenance of the ER lipid distribution is regulated by the presence of ATP-dependent lipid transporters, called flippases and floppases. Additionally, proteins called scramblases are believed to be involved in an ATP-independent lipid-transport process (34). The ER contains low amounts of negatively charged lipids in the cytosolic membrane leaflet. Thus, it is characterized as a membrane with loose lipid packing and low electric charge together with a symmetric lipid distribution (35). The ER network extends throughout the cytoplasm and is highly dynamic (36). With the use of electron microscopy the ER sheets, called rough ER, show a high number of membrane bound ribosomes, while the tubular ER membrane, called smooth ER, show much less ribosomes (Fig 8) (33).
15
A B
Fig. 8: Structural images of the ER subdomains. A. Image of a thin-section electron micrograph of ribosome-covered ER sheets in secretory cells of silk glands from a silkworm. B. Image of a scanning electron micrograph of the ER tubular structure from rat white skeletal muscle fibers. Both images are from (33).
Synthesis and processing of membrane and secretory proteins occur in the rough ER (37). At the smooth ER, protein packaging and transport to the Golgi apparatus and further to the plasma membrane takes place (31, 38).
16 4 Proteins inside or at the surface of the membrane
About 50 % of the mass of biological membranes of most animal cells consists of lipids, with the rest being membrane proteins. The lipid bilayers provide many functions such as acting as selective permeability barriers, transporting matter between the interior and the exterior of the cell, and assisting in various catalytic processes. These specific tasks are performed by membrane proteins (39). Of all protein-encoding genes in most organisms, 15 to 30 % account for integral membrane proteins. Yet membrane proteins represent less than 1 % of all structures in the Protein Data Bank (40-42). Membrane proteins display chemically different features simultaneously. The non-polar acyl chains of phospholipids and sterols in the lipid bilayer core surround the membrane-embedded section of a membrane protein. Hydrogen bonds cannot form between the carbonyl oxygen or the amide nitrogen atoms of the polypeptide backbone and the hydrophobic lipid tails. As an effect, intramolecular hydrogen bonds help stabilize membrane proteins (43, 44). The phospholipid head groups at the membrane interface accounts for another feature of the immediate environment of the membrane protein. Dipole-dipole interactions and hydrogen bonds are formed between the protein and the lipid head groups (45). Finally, membrane proteins can contain extra-membraneous domains that protrude into the aqueous phase from both the inner and outer surfaces of the membrane (43). Membrane proteins differ widely in structure and their way of connecting to the membrane. Roughly speaking, membrane proteins can be divided into two classes: peripheral and integral. The peripheral membrane proteins do not span the hydrophobic core of the lipid bilayer. Instead they adhere to either face of the membrane by electrostatic and hydrophobic interactions, or via non-covalent interactions with other membrane proteins. With relatively mild extraction methods, such as solutions with a high pH or salt concentration, these proteins can be released from the membrane (1). Bilayer-spanning integral membrane proteins (IMP) are permanently anchored in the membrane. In contrast to the peripheral membrane proteins, this group of proteins requires harsh extraction methods, such as exposure to
17
4.1 ȕ-barrel membrane proteins
ȕ-barrel membrane proteins are typically found in the outer membrane of gram-negative bacteria, and in mitochondrial and chloroplast outer membrane (53). Proteins in the outer membrane of gram-negative bacteria called porins function as channels through which small solutes can passively diffuse across the bilayer. Every second K\GURSKRELFUHVLGXHLQHDFKȕ- strand faces toward the surrounding lipid environment. The protein assembles into a cylinder-OLNHVKDSHIRUPLQJDQDQWLSDUDOOHOȕ-barrel (54-56) Fig 5B. This type of conformation allows for intramolecular bonds between WKHEDFNERQHK\GURJHQERQGGRQRUVDQGDFFHSWRUVLQQHLJKERULQJȕ-strands. T\SLFDOO\ȕ-barrels are made of an even number of tilted strands, with the minimal number of strands being 8 (57, 58). ȕ-barrel proteins are made in the cytoplasm. The unfolded peptide translocates through the Sec translocon machinery found in the bacterial LQQHUPHPEUDQH:LWKWKHVXSSRUWRIWKH%DPFRPSOH[WKHȕ-barrel proteins get assembled and integrated into the outer membrane (59). $IWHUUHDFKLQJWKHLUGHVWLQDWLRQȕ-barrels function in a number of different cellular processes such as transport of fatty acids, sugars and nucleosides, in signal transduction and in the permeation of host cell membranes by toxins, to mention a few (58, 60).
4.2 Į-helical membrane proteins
In the absence of water molecules in core of the membrane, no hydrogen bonds can form between the water and the backbone carbonyl oxygens and amide nitrogens in polypeptide chain. Instead, hydrogen bonds from internally, leading to the formation of transmHPEUDQHĮ-helices (61) Fig 5A. Genomic analyses show that 20 to 30 % of all open reading frames in many RUJDQLVPVFRGHIRUĮ-helical membrane proteins (42). 90-95 % of all integral membrane proteins haYHEHHQSUHGLFWHGWRIRUPDQĮ-helical membrane protein structure, with the rest forming ȕ -barrels. (62). $OWKRXJKĮ-helical membrane proteins differ in shape and size, their most obvious characteristic is stretches of 20-25 hydrophobic amino-acid residues. These stretches form transmembrane Į-helices that span the entire membrane, often with a more or less pronounced tilt (63, 64). 7UDQVPHPEUDQHĮ-helices can be anything from 15 to 40 residues long (65). During synthesis, as the polypeptide chain translocates through the WUDQVORFRQWKHK\GURSKRELFVLGHFKDLQVRIWKHWUDQVPHPEUDQHĮ-helices partition into the surrounding lipid environment, concomitant with the formation of backbone hydrogen bonds. Together, these two energetically favorable procedures overcome the unfavorable entropy of folding the
19 polypeptide backbone(66, 67). Į-helical membrane proteins are as diverse in their structure as in their function even though they share a common structural unit, namely the WUDQVPHPEUDQHĮ-helix. Proteins are known that have more than 30 WUDQVPHPEUDQHĮ-helixes in a single polypeptide chain. Į-helical membrane proteins are prone to interact with each other, forming large protein complexes. One such example is the 13-subunit cytochrome c oxidase situated in the inner mitochondrial membrane (68, 69). Į-helical membrane proteins are found in all different membranes across all kingdoms of life. With their high functional diversity, their biological importance cannot be overestimated. Their functional complexity is further elaborated by the formation of homo- or hetero-oligomeric structures (62). G protein-coupled receptors coQVWLWXWHDODUJHFODVVRIĮ-helical membrane proteins. Their physiological roles involve signal transduction in vision, olfaction, ATP synthase, aquaporins and ion channels for water and ion homeostasis modulation respectively (50). ,QSURNDU\RWHVPRVWĮ-helical membrane proteins are present in the inner membrane. However the polysaccharide transporter Wza has been found, in the form of a transmembrane helix bundle, located in the outer membrane of E. coli (70).
4.3 Protein-lipid interactions
The lipid matrix of the membrane provides a site for protein-lipid interactions. Proteins and lipids perform different cellular processes together, such as intracellular trafficking, cell division and the formation of organized patches or “rafts” in the membrane - microenvironments where signal transduction and membrane transport can take place. The importance of the interplay between proteins and lipids is reflected in the process during which a membrane protein is folded to its final structure in order to function properly (71). Protein-lipid interactions both annular lipids in a protein’s immediate surroundings and lipids buried in protein surface cavities or between protein subunits in membrane protein complexes (71). The cylindrically shaped phosphatidylcholine (PC) corresponds to more than half of the phospholipids in the majority of eukaryotic membranes, and promotes bilayer formation. However, when PC is mixed with non-bilayer forming lipids such as phosphatidylethanolamine and cardiolipin, shape mismatch induces curvature elasticity in the membrane. This can have an impact on the process of membrane protein insertion and folding (20, 72). The bacterial SecYEG translocon machinery has been seen to function optimally when surrounded by the lipid mixture consisting of anionic phosphatidylglycerol and non-bilayer lipids (73).
20 To function properly, just about all membrane proteins require that the surrounding membrane has certain properties in terms of, e.g., fluidity, thickness or lateral surface pressure (74). On the other hand, during their lifetime, membrane proteins adapt to major membrane environmental changes and thus can reside in different lipid surroundings. The hydrophobic membrane protein surface is often broader or thinner compared to the lipid bilayer core. This is referred to as hydrophobic mismatch. Membrane proteins adapt to this situation by aggregation, helix tilting or oligomerization in order to avoid exposing their hydrophobic surfaces to the surrounding hydrophilic milieu. In addition, the lipid bilayer can get disrupted to reform its shape to match the protein structure. As a result of this event, acyl chains get stretched and a reassembled lipid phase is formed (75, 76). Transmembrane helices of proteins embedded in the plasma membrane are in general longer than those found in proteins localized to the Golgi complex and ER membrane. Thus, hydrophobic mismatch affects membrane protein function, sorting and organelle localization (77). Finally, the sterol content in various membranes has been observed to influence the sorting of proteins in the secretory pathway (78).
21 5 Protein biogenesis
The life of all proteins starts in the ribosome, located in the cytosol of eukaryotic or prokaryotic cells. Membrane proteins need to be transported to their target membrane. To enable this, specific protein targeting routes have evolved. Membrane proteins get recognized and delivered to protein transport channels through which they are translocated across or inserted into membranes, in most cases while they are being synthesized (i.e., co- translationally). The most widely used protein translocation machinery is composed of an evolutionary conserved membrane-embedded secretory (Sec) system. This protein-conducting channel handles protein transport through both co- and post-translational translocation pathways in prokaryotes (the SecYEG complex) and eukaryotes (the Sec61 complex) (79).
5.1 Protein synthesis within the ribosome
Ribosomes are large, complex cellular machines and represent around 25 % of the cell’s dry weight. Both the bacterial and eukaryotic ribosomes are composed of two subunits (Fig. 10). The subunits are made of multiple proteins and RNA molecules. The bacterial subunits have 30S and 50S sedimentation coefficients, respectively, whereas the eukaryotic units have on average 40S and 60S. The eukaryotic (excluding chloroplast and mitochondria) ribosomes are both larger and more complex than the bacterial ribosomes. Nevertheless, the gross composition of the different kinds of ribosomes are strikingly similar (46).
22
6 Protein targeting and transport
Correct targeting of newly synthesized proteins to their final destination in the cell is a key requirement in order for the protein to function properly. Intrinsic signals of the proteins direct them to their target cellular or subcellular locations (6, 10, 82, 83). A smaller set of proteins encoded by the chloroplast and mitochondrial DNA are synthesized by organellar translation machineries (84).
6.1 Signal sequence
Signal sequences function as “address labels” on proteins that are to be delivered to a destination, which can be, e.g., the ER, mitochondria or cell exterior. Translocation machineries assist the proteins during translocation through the membrane and help to insert hydrophobic protein segments into the membrane (8). Such targeting signals are primarily found as N-terminal extensions of polypeptide chains. The structural and chemical properties of the amino acids making up the signal sequence influence the journey of the protein. Characteristics such as hydrophobicity, length and distribution of charged residues determine the final destination (82, 85, 86). Proteins travelling from the cytoplasm destined to end up in the mitochondria have 15 to 55 residue long targeting sequences named presequences. These sequences contain a high number of positively charged amino acids and a propensity to form amphiphilLFĮ-helices (82, 87). The TOM complex (translocase of the outer membrane) functions as a molecular gatekeeper for both soluble and membrane proteins travelling across the outer mitochondrial membrane and into the intermembrane space. Further, the TIM complex (translocase of the inner membrane) function as a gateway through which proteins containing a presequence are subsequently passing into the mitochondrial matrix or inserted into the inner mitochondrial membrane (88). The presequences are finally processed by proteolytic removal carried out by specific peptidases (89). ER signal sequences have a more hydrophobic character than mitochondrial presequences. They contain a 7 to 13 residue core K\GURSKRELFVWUHWFKZLWKDWHQGHQF\WRIRUPDQĮ-helix, flanked by a
24 positively charged N-terminal domain and a polar C-terminal domain (82, 90). Secretory proteins targeted for export from the cell, as well as proteins targeted to insert into the Golgi, ER or plasma membrane, are all first routed to the ER. The signal sequence of secretory proteins often has a cleavage site downstream of the hydrophobic domain. The positive charges at the N- terminal domain induce a Nin/Cout orientation of the signal peptide. Signal peptidases further process the cleavage site located on the luminal side of the ER membrane (91, 92). In the case when there is no available cleavable signal sequence, the first hydrophobic transmembrane segment of a membrane protein takes over the targeting function and also firmly attaches the protein in the membrane of the ER (93). The favored protein targeting pathway is influenced by the overall hydrophobicity of the signal sequence. Highly and moderately hydrophobic sequences are directed to the ER translocon in a co-translational fashion, whereas most nuclear-encoded mitochondrial proteins follow a post-translational translocation route (85, 86). Randomly generated sequences of the right composition have been shown to be able to function as signal sequences, targeting proteins to the ER, the bacterial inner membrane, and mitochondria (8). In the 1960s, Palade paved the way for this field of study by his research on the secretory pathway in eukaryotes (94). In 1971 Sabatini and Blobel suggested the “signal hypothesis” to explain co-translational translocation of ribosome-nascent chain complexes directed to the ER membrane (7). Finally, Blobel and Dobberstein proposed in 1975 that membrane proteins could form an aqueous pore through which secretory proteins could move through the ER membrane (6).
6.2 Protein targeting to the bacterial inner membrane and the ER
In bacteria, the majority of inner membrane proteins follow a co- translational insertion pathway, while proteins destined to be secreted across the inner membrane utilize a post-translational translocation pathway (95). Protein targeting to the ER membrane in mammals is in general co- translational, with a small number of secreted proteins translocating in a post-translational fashion. In yeast, both kinds of translocation routes are used (94, 96).
25 final step in the co-translational translocation pathway is the disassembly of the SRP-SR complex by GTP hydrolysis of the complex components, allowing them to enter a new round of the “SRP-cycle” (108). In addition to the Sec-type translocon pathways, there is a remarkable number of different mechanisms for protein export. In bacteria these systems count up to sixteen and can manage protein secretion, sorting and membrane integration. Out of all translocation machineries, the Sec route is ubiquitous and essential throughout all three kingdoms of life, where the co- translational pathway mostly handles membrane protein targeting and insertion (109). Protein targeting is not carried out in a spontaneous manner, and different energetic conditions dictate protein translocation into or through the bacterial and ER membranes. In bacteria, one driving force for protein export is provided by the ATP-dependent SecA protein, which empowers the movement of unfolded proteins across the inner membrane (110, 111). When it is located in the cytoplasm it binds preproteins, and when it is at close proximity to the membrane, it associates with the translocon channel. The proton motive force across the inner bacterial membrane is another driving force inducing translocation of proteins, and can stimulate the movement of amino acids carrying a negatively charge across the membrane (112). In eukaryotes, the driving force for co-translational translocation across the ER membrane is provided by the ribosome. However, some studies indicate that an additional component such as the soluble chaperone, Bip, found in the ER lumen, may assist in optimizing the translocation process (113).
6.2.2 Post-translational protein targeting pathway
Observations have reveled alternative protein translocation routes, including post-translational translocation pathways. Some proteins are completely translated by the ribosome without any SRP-mediated targeting during the process. Only after this step, they proceed to translocate through the SecY/Sec61 translocon channel, in a post-translational mode of translocation. The majority of proteins that are destined for this route are soluble proteins, such as secretory proteins and less hydrophobic membrane proteins (85). Proteins undergo different mechanisms of post-translational translocation in eukaryotes and prokaryotes. In eukaryotes, cytosolic chaperones bind the unfolded polypeptide, after it has been released from the ribosome, in order to prevent aggregation. The polypeptide then binds to the Sec61 channel, leading to translocon-mediated translocation and chaperone dissociation from the substrate (Fig. 12).
27
7 Protein translocation machineries
Translocons function as major facilitators in the mechanism of targeting membrane and soluble proteins to various membranes and subcellular compartments. They also manage to handle stress and developmental cues. Furthermore, they are involved in both targeting and maturation events of proteins, i.e., processes involved in protein folding, assembly and modifications (8). Substrates aimed for translocation dock at specific sites on the translocon with their targeting signals. The translocon coupled with a translocation driving force, mediates protein export in a selectively permeable and conducting manner. The processing and folding of most proteins progress at their destination compartment. However, with their different pore sizes, some translocons can manage to accommodate and handle the translocation of completely folded proteins. One such example is the Twin-Arginine Trasnlocation (TAT) translocon that translocates folded proteins carrying a twin-arginine motif in their signal peptide. Folded proteins can be translocated across the thylakoid membrane, peroxisomes, the nucleus and the inner membrane of bacteria (114-120).
7.1 The Sec translocon
The eukaryotic Sec61-translocon is located in the ER membrane. It helps secretory proteins and membrane proteins translocate across and integrate into the membrane, respectively. The homologous eubacterial SecYEG and archaeal 6HF<(ȕtranslocons are both located in the plasma (inner) membrane where they also function as protein-conducting channels. The Sec61-translocon is a heterotrLPHUEXLOWRIĮ-ȕ- DQGȖ-subunits. The Į- and Ȗ-subunits are conserved through evolution and are necessary for cell VXUYLYDO7KHVHTXHQFHRIWKHĮ-VXEXQLWLVYHU\VLPLODUWRWKDWRI6HF<WKHȕ- subunit is similar to the bacterial SecG and that of the Ȗ-subunit to SecE from bacteria. In 2004 a crystal structure was resolved for Methanococcus jannaschii SecYEG, revealing structural details of the Sec complex. With its 70KHOLFHVWKHĮ-subunit is the major component of the channel. It has an hourglass-form, when looked at from the side, together with a constriction ring built by hydrophobic amino acids (121). The pore allows loops of
29 membrane proteins and secretory proteins to pass through the membrane (122). The size of the pore is still rather controversial and it is thought to be somewhat flexible and adapt to nascent chains passing through the channel (123). Viewed from the top, TM helix 1-5 and TM helix 6-RIWKH6HFĮ- subunit appear as a clamshell. These two halves have an interconnecting hinge, making it possible for a lateral gate located between TM2b and TM7 to open up to the lipid bilayer. The translocon cavity facing the cytosol is found as empty, whereas, the cavity facing the extracellular compartment obstructed by a plug in the closed form of the channel. (121). The lateral gate of the channel lets TM segments partition into the hydrophobic milieu of the lipid bilayer. A clear mechanism has still not been established for this cellular process. However, it is thought that the presence of a hydrophobic peptide (such as a signal peptide) localized in the lateral gate can stabilize an open gate conformation (124). Studies have also revealed interactions between the signal sequences and TM helices 2 and 7 (125). In addition, recent structural research has shown that in the presence of a transmembrane segment in the lateral gate, TM helices 2 and 7 move DSDUWDQGVHSDUDWHWKHWZRKDOYHVLQWKHĮ-subunit with about 12 Å (126). 7KHUROHRI6HFȖ 6HF( LQWKHWUDQVORFRQLVWRFODPSWKHKDOYHVRIWKHĮ- subunits toJHWKHUWKXVVWDELOL]LQJWKHVWUXFWXUH6HFȕ 6HF* LVQRQ-essential and is located on WKHSHULSKHU\RIWKHĮ-subunit. Its function has not yet been clearly established. However, it has been speculated that it can facilitate co- translational translocation (121, 127) (Fig. 14). The evolutionary conservation from the SecY translocon to mammalian Sec61 translocon has been confirmed by Cryo-EM structures (128-130).
A B
Fig. 14: The SecYEE complex crystal structure from Methanocaldococcus jannaschii. Image A shows a top cytosolic view side and B shows a side view. The blue TM2b and yellow TM7 form the lateral gate where the TM partitions into the cell membrane. The plug is colored in green. Images are adapted from (121).
30 7.2 Accessory components
A number of proteins have been discovered that are directly or indirectly associated with the Sec translocon, and thereby influence the protein translocation process (93).
7.2.1 Bacterial SecDFYajC and YidC
The membrane protein complex SecDFYajC interacts in a temporary manner with the Sec translocon channel in bacterial cells. Inactivation of SecD and SecF in E.coli results in growth and protein secretion abnormalities. However they are not considered as essential proteins for cell viability (131). On the other hand, YidC has an essential function in bacteria regarding membrane insertion of a subset of membrane proteins (132). Studies of crosslinking with YidC indicate how it associates in a transient fashion with the SecYEG complex as membrane proteins are being inserted into a membrane. During the interacting phase of YidC with SecYEG, the accessory proteins SecDF may also be involved (133). YidC also functions as an insertase by itself, where most of its substrates are membrane subunits of respiratory complexes (134). However, the majority of membrane proteins rely on YidC for its folding capacity rather than its membrane insertion functionality. F1FO-ATPase is an example of a protein complex that is folded by YidC (135, 136).
7.2.2 Eukaryotic TRAM, Calnexin, Calreticulin, OST, PDI
The translocation-associated membrane protein (TRAM) is a component belonging to the translocon in mammalian cells. The core units of the co- translational translocon machinery consist of TRAM and the heterotrimeric 6HFĮȕȖ:KHQFR-expressed in vitro they show an adequate level of translocation activity (137). In all eukaryotes and in a few of prokaryotes the protein-modifying process of N-linked glycosylation takes place (138). In eukaryotes, the process is carried out by the membrane protein complex oligosaccharyl transferase (OST) (Fig 15). It is localized in the near proximity of the translocon and can interact with
31
7.3 Signal peptidase
Proteins destined to the cell surface usually have a cleavable signal sequence at their N-terminal end. This is cleaved by type I signal peptidase (SPase), identified in bacteria, ER and mitochondria. The catalytic domain is located on the luminal (periplasmic in bacteria) side of the membrane. One to two transmembrane segments at the N-terminal end of the protein anchor the SPase to the membrane (153, 154). The SPases are serine-lysine proteases, except for the ones located in the ER (155). Ser-90 in E. coli SPase is an active site residue, which is conserved in the homologous ER SPase complex. Compared with the bacterial SPases, Organellar SPases have replaced a catalytic Lys with a His residue. The ER SPases contain either a serine-histidine dyad or a serine-histidine-aspartic acid triad (156). Both SPases and OST complexes seem to be close to the translocon, where they can interact with translocating nascent chains. There is roughly an equal number of SPases, ribophorin I and ribosomes present in rough ER microsomes. Thus, probably one OST complex and one SPase are bound to each translocon (113).
33 8 ,QWHJUDWLRQRIĮ-helical membrane proteins into the membrane
The Sec translocon participates in the identification, orientation, lateral WUDQVSRVLWLRQDQGSDUWLWLRQLQJRIĮ-helices into the lipid bilayer (157). A transmembrane region in a nascent protein chain can initially be recognized already in the ribosome (158, 159). The sequence hydrophobicity is the main driving force for the integration into the lipid bilayer (160). Crosslinking experiments (161) show that a certain site in the translocon channel allows for transmembrane segments to interact with the hydrophobic domain of the lipid bilayer. The transmembrane segment is thought to equilibrate dynamically between the translocon and the membrane. This goes in line with the biological hydrophobicity scale, which is discussed in detail later in this thesis (162, 163). Junne et al have analyzed the pore of the translocon, where they introduced substitutions in the ring of the channel pore. They observed that as the pore changed into a more polar or charged character, it could promote membrane integration of less hydrophobic transmembrane helices. Thus, the reduced hydrophobicity of the pore alters the equilibrium between the translocon pore and the lipid bilayer (164). Hydrophobic segments have in addition been proposed to induce the lateral gate to open for membrane integration of the transmembrane helix (124). The eighth transmembrane segment of the CFTR protein has been identified to be retained in the Sec61 translocon with an inhibited membrane integration upon depletion of ATP molecules. This may indicate that the translocon has an active role in helix-integration (165).
34
The high energetic cost for the presence of polar and charged residues in the transmembrane segment, is to some degree compensated by salt bridges between helical segments (174). The electrostatic and van der Waals interactions between helices have been profoundly researched, e.g. Asn-Asn or Asp-Asp interactions have been found to stabilize transmembrane helix-helix interactions (175, 176).
9.2 Proline and glycine in transmembrane segments
The proline residue stands out from the rest of the 19 amino acids, since its amine nitrogen is included in the ring structure that is connected to two alkyl groups. With its rigid structure, it breaks up and kinks an Į-helix (177), and triggers helical hairpin formation in long enough hydrophobic domains of the protein. Proline-induced kinks in membrane proteins may carry an important role in structural stability and function (178-180). Prolines are more common in transmembrane helices, compared to Į-helices of globular proteins (181). Sequences with a high number of prolines and glycines can more easily form coils, leading to increased structural flexibility and resulting in swivels, hinges (180, 182) and reentrant domains (65, 183, 184). They often occur in channels and transporters for efficient functioning (184).
9.3 The “aromatic belt”
Tyr and Trp are two aromatic amino acids that are often found at the ends of TM helices. Given their amphipathic nature, they can interact through hydrogen bonds with lipid head groups and make hydrophobic interactions with the lipid acyl chains. This may give stability to the helix relative to the bilayer. On the other hand, these two amino acids can destabilize a helix while residing in its central part. In addition to Tyr and Trp, another aromatic and entirely hydrophobic amino acid is Phe. It is enriched in the central part, rather than at the ends of transmembrane helices (43, 45, 172, 185, 186).
9.4 “Snorkeling”
From high-resolution protein structures, many charged amino acids and secondary protein structures with an irregular form have been identified in the core of the hydrophobic region of membrane proteins (162, 163, 183). Charged amino acids found in the membrane core are energetically
36 unfavorable. The positively charged Lys and Arg have long aliphatic side chains that allow the charged moiety to “snorkel” up to the lipid head groups in the interfacial part of the membrane (45, 186, 187). The side chain of Asp is however too short and cannot “snorkel”. Repulsion may occur between residues with a negative charge and the negative charge at the head group of phospholipids in the membrane (188). The “snorkeling” mechanism may help polar residues to be tolerated in transmembrane helices (187). Simulations indicate that this behavior enable polar side chains to form a micromilieu with attached water molecules in the membrane core (189). Residues with a positive charge in close proximity to transmembrane helices strongly influence the topology of proteins. Acidic residues have much less potency in directing the protein topology. In addition, they present no statistical preference for localization in loops on either side of the membrane (168, 190). However, their presence have been found to affect topology determination under special conditions where high numbers of negative charged residues are present or are positioned close to marginally hydrophobic transmembrane segments (191-193).
37 10 Membrane protein topology
The topology of membrane proteins, i.e. the number of transmembrane helices, their location in the protein and their orientation relative to the membrane, is important knowledge for the study of membrane protein structure and function. The majority of proteins have a unique membrane topology, and homologs share the same orientation. In prokaryotes a small number of proteins have dual orientations within the membrane. The E. coli inner membrane protein, EmrE, which is a small multi-drug transporter, is an example of such a protein (194-196). This is a small, membrane protein with four transmembrane segments. Usually this type of proteins have very few amino acids with a positive charge (see below), thus the charge bias of the loops located externally and internally is small (197, 198). The charge bias of EmrE can be altered by adding an amino acid with a positive charge into one of the loops, even in the situation where the residue is at the C-terminal end of the protein (198). Topology predictions can be made with the use of bioinformatics tools (199- 201). with a success rate estimated at 65-70%. By combining multiple bioinformatics programs, the accuracy in topology prediction can be increased (199, 201). The transmembrane segments of membrane proteins can obtain different topologies in the membrane (Fig. 17). Membrane proteins containing only one single-spanning transmembrane segment have a Nexo/Ccyt or Ncyt/Cexo orientation in the lipid bilayer. Membrane proteins in the type I class (Nexo/Ccyt) carry a cleavable signal sequence that initially directs the protein to the bacterial inner membrane or the eukaryotic ER membrane. Membrane proteins in the type II class (Ncyt/Cexo) lack a cleavable signal sequences. Instead, they carry a signal-anchor sequence and the C-terminal end is induced to translocate to the exoplasmic side of the membrane. In the type III class (Nexo/Ccyt), the opposite topology orientation process occurs: the protein possesses a reverse signal-anchor sequence and the N-teminal domain orients itself across the membrane (202).
38 positively charged amino acids. The topology of the membrane protein Lep in E. coli, having two transmembrane segments, can be altered by substitution of positively charged amino acids. However, by adding or changing negatively charged residues in the protein, results show less dramatic topology alterations compared to positive charges. Sequence studies from a large set of different organisms shown no strong biased distribution of residues with a negative charge. Within the Sec61 translocon, some charged amino acids assist in orienting a signal sequence into an Ncyt-topology (216). The composition of lipids in the membrane also affects the orientation of transmembrane segments. Interactions might take place between the positively charged amino acids and the lipid head groups bearing a negative charge (188). In addition, the lipid mixture in a membrane can have an effect on the selectivity for negative charges and thus influence membrane protein topology (217). The electrochemical potential across the prokaryotic plasma membrane is probably also a contributing to the “positive-inside rule” (218).
10.2 Lipids as topology determinants
The zwitterionic lipid phosphatidylethanolamine (PE) has been shown to act as an important component during the folding and topology orientation process of membrane proteins. Membrane integration of the transmembrane segments of the membrane protein LacY in E. coli has been studied under conditions where PE is absent from the inner membrane, and it was found that the six N-terminal transmembrane helices inserted into the membrane with an inverted topologywhile the seventh helix failed to insert (217).
10.3 Other topology determinants
The hydrophobicity of signal sequences has been shown to have an impact on protein topology in the membrane. The Nexo/Ccyt orientation tends to be a favored topology for transmembrane segments with a more hydrophobic character. In addition, the length of the segment also influences the topology. As the length of the segments increases, the fraction of proteins with the Nexo/Ccyt topology also increases (219). In eukaryotic cells, the N-terminal domain can undergo covalent modification, e.g. glycosylation, where an oligosaccharide is attached to the protein. This event inhibit a possible re-orientation or backsliding of a transmembrane segment, thus fixing the N-terminal end of the protein to the lumenal side (220).
40 10.4 Hydrophobic mismatch
Hydrophobic mismatch may trigger both helix-helix interaction (221), tilting of helices in the lipid bilayer (222) and formation of kinks within a helix (223).
10.5 Re-entrant regions
Several membrane proteins contain short segments named re-entrance regions. These segments enter and re-enter the membrane from the same side, i.e., they do not fully span the lipid bilayer. They mostly appear in channels and transporters such as aquaporin 1 (224), the glutamate transporter GltT (225) and the NCX1 Na+/Ca2+ exchanger (226). More than 10 % of membrane proteins carry this type of regions. Water and ion channels are typical proteins where these regions have been found, whereas, they are least common in surface receptors. Re-entrance regions penetrate between transmembrane helices, and are thus shielded from contact with lipids (184). Re-entrant regions contain a high number of small residues, typically glycine (227).
10.6 Topology mapping
The hydrophobic transmembrane segments in multispanning membrane proteins traverse the lipid bilayer in a zigzag manner. The exposed loops are either oriented into the cytoplasm or the exoplasm, which are isolated from each other by the membrane. In order to experimentally establish the topology of proteins, the hydrophobic domains of the protein must be confirmed as transmembrane segments and the loops need to be mapped to either one of the two mentioned compartments (228). This can be achieved in various ways, e.g., by analyzing fusions between a membrane protein and topology reporters such as PhoA and GFP, or by glycosylation mapping.
10.6.1 PhoA
A membrane protein topology reporter that has been widely utilized in E. coli is PhoA. It was one of the first proteins used in an assay to map protein topology by identifying protein loops oriented into the bacterial periplasm (229). In order for PhoA to become cacalytically active, two disulfide bonds need to be formed. These bonds can form only if the PhoA protein enters the
41 periplasm, whereas PhoA folds improperly when situated in the cytoplasm. The active form of PhoA can catalyze the hydrolysis of chromogenic substrates, affording a simple way to assay its activity in vivo and hence determine its cytoplasmic/periplasmic orientation when fused to different loops in an inner membrane protein (230).
10.6.2 GFP
The 26.9 kDa green fluorescent protein (GFP) is derived from the jellyfish Aequorea victoria (231). It is a common cytoplasmic reporter tool when engineered to the C-termini of proteins (232, 233). Cytoplasmically located GFP can fold and become fluorescent, while it does not fold and remains non-fluorescent when located in the periplasm (234).
10.6.3 Glycosylation mapping
Topology mapping of proteins can be carried out by introducing N- glycosylation acceptor sites at certain locations in the protein of interest by the use of site-directed mutagenesis. The addition of an oligosaccharide on the acceptor site can only take place in the same cellular compartment as the active site of the OST complex is located in, namely the eukaryotic ER lumen. When a target site in a protein becomes glycosylated, the molecular mass of the protein increases by approximately 2 kDa, which can be detected by SDS-PAGE (235).
10.6.4 Protease-protection assay
For topology determination of a membrane protein, the proteases K and trypsin can be used by addition to eukaryotic microsomes or bacterial spheroplasts. The role of these enzymes is to degrade parts of the protein that are exposed on the outside of the membrane. The unexposed protein domains that are positioned in the eukaryotic ER lumen or bacterial cytoplasm remain undegraded and can be visualized by SDS-PAGE (228).
10.6.5 Epitope tags
Epitope tagging can be applied by which a protein of interest is engineered to become immunoreactive against a known antibody. Anti-tag antibodies have high species-specificity and assists in detecting the protein of interest. Thus, the need for protein-specific antibodies is unnecessary. The frequently
42 applied epitope tags in experimental work include: cMyc, glutathione-S- transferase (GST), FLAG, maltose binding protein (MBP), hexahistidine (6X-His), GAL4, GFP and ȕ-galactosidase (236).
10.7 Topology predictions
The development and use of programs dealing with protein topology prediction has helped in the study of membrane protein structure. The only requirement is the input of a known protein sequence. However, with the use of the prediction program, comes also its disadvantage in the form of incorrect topology prediction. The early programs managed simply to predict the occurrence of transmembrane helices within proteins and their localization (237, 238). In a later stage, as the positive inside rule was defined and included in the prediction algorithms, it became possible to predict both the number of transmembrane helices and their orientation in the lipid bilayer (239). Nowadays, improved programs can include additional membrane protein structural information such as prediction of Į-helix or ȕ-sheet conformations, presence of organellar localization sequences and re-entrant protein domains (51). The updated predictors are set up in the form that experimental data can be combined with protein predictions. This results in an increased prediction reliability. Despite the improved modern programs, still certain protein domains such as the re-entrant regions and helical hairpins are difficult to predict (240).
10.7.1 Hydrophobicity scales
Studies by Hessa et al describe a biological hydrophobicity scale where the side chains of different amino acids have different affinity for the membrane (162, 163). The scale is based on quantitative measurements of the ER membrane insertion efficiency of model transmembrane segments. The laboratory-engineered model transmembrane segments, also named H segments, were built on a 19 residues long Ala and Leu stretch. The segment was flanked on each side by two glycosylation acceptor sites in order to function as a membrane insertion reporter assay (241). Ala and Leu residues favors the formation of Į-helices and are frequent in natural transmembrane helices (242). The length of a 19 residue helix is approximately 30 Å. This is in line with the thickness length of the membrane’s hydrophobic section. Leader peptidase (Lep) has two natural transmembrane segments. and inserts into the ER membrane with both the N and C terminus in the ER lumen. H segments were cloned into Lep with the aim to function as a third transmembrane helix (243). Lep variants composed of the three
43 transmembrane segments were expressed in vitro in the presence of ER- derived dog pancreas rough microsomes to study the ER insertion of the H segment (241). An apparent free energy of membrane insertion (ǻGapp) was calculated on the basis of the H segment membrane insertion efficiency. The propensity of membrane insertion for all 20 naturally occurring amino acids was determined adding each residue to the H segment (Fig. 18) (162, 163).
Fig. 18: The “biological” hydrophobic scale gives the contribution to the free energy of membrane insertion of the H-segment for each of the 20 natural amino acids. The graph is adapted from (162).
The biological hydrophobicity scale matches well with the Wimley-White water/octanol scale (244). By moving each studied residue along the H segment free energy insertion profiles were measured. The results were in line with the physiochemical nature of a lipid bilayer. Therefore, both side-chain chemical properties and their localization within the transmembrane helix influence the membrane insertion process. Further, in order for membrane insertion to take place, a transmembrane segment must acquire a helical structure, as suggested by the observation that the addition of a Pro residue in the center of the H segment results in high free energy values. Transmembrane segments appear to partition between the polar Sec61 channel and the apolar membrane milieu (161). As the translocon rearranges into its open state, it allows the translocating peptide to move to the translocon-lipid bilayer interface (245, 246). The same type of “biological” hydrophobicity scales have been measured for different organisms. While broadly similar, some differences were found between the scales. Half-maximal insertion was seen at an H segment
44 composition of 16 Ala + 3 Leu for in vitro expression in the mammalian ER membrane (162, 163), at 15 Ala + 4 Leu for in vivo expression in yeast (247), and at 17 Ala + 2 Leu for YidC-mediated bacterial inner membrane insertion (248). The “biological” hydrophobicity scale derived from the ER mammalian expression system, is available at the web server http://dgpred.cbr.su.se/. The server provides predicted ǻGapp values for membrane insertion of protein sequences. By scanning protein sequences that are naturally occurring through the predictor server, free energies of membrane insertion for mammalian single spanning membrane proteins and soluble proteins have been recorded, showing negative and positive ǻGapp values, as expected. Membrane insertion predictions of multi-spanning membrane proteins with a defined three-dimensional structure, show that approximately 25% of the transmembrane segments of the proteins have ǻGapp values > 0 kcal/mol, indicative of an unfavorable membrane insertion process. This suggest that some transmembrane segments in multi-spanning membrane proteins need to rely on interactions with neigbouring segments for membrane insertion (249).
45 11 Folding RIĮ-helical membrane proteins in the membrane
Ever since the pioneering work of Anfinsen, which demonstrated that RNaseA could be re-folded from a denatured state (250), extensive research has been focused on protein folding mechanisms of both water-soluble proteins (251-253) and integral membrane proteins (75, 254, 255). However, studies of integral membrane proteins have not progressed to the same extent as the investigations on the water-soluble proteins, due to a number of reasons (75, 256). The surface properties differ between membrane proteins and water-soluble proteins. In addition, elastic properties and lateral forces in the environment of membrane proteins influence their membrane orientation and function (255). Therefore, when studying molecular structure and function of membrane proteins a key factor is to manage to mimic the native lipid bilayer milieu that maintain the folded and functional form of the protein. This has proven to be a major barrier in membrane protein folding investigations, thus lowering the pace of research advancements in this field (256). Data from both kinetic and thermodynamic analyses are important in order to be able to acquire a full understanding of the folding mechanism. One approach to measure the free energy of the folding process is by taking advantage of a reversible chemical denaturation processes. From a microscopically reversible two-state system, a folding equilibrium constant is possible to obtain. With this, the change in free energy in relation to the reaction can be calculated. When analyzing water-soluble proteins, the relationship between the concentration of the denaturant and the free energy is mostly linear. Membrane protein folding studies are often plagued by protein aggregations and irreversible denaturation. For a handful of membrane proteins, however, reversible folding work on membrane proteins have proven successful, however, folding reactions can run through a two-state transition where the relationship between the denaturant and the free energy appears in a linear manner (66, 256-261). As an example, the Į- helical membrane protein, bacteriorhodopsin, has been identified to fold in a microscopically reversible process, undergoing a two-state transition (262). As the work in the field of in vitro folding is advancing, the recognition of chaperones as key players in assisting protein folding in vivo has emerged (263). Investigations have recently begun to focus on cellular complexities in the folding landscapes, e.g., co-translational folding, importance of protein
46 degradation and the influence of the high concentration of macromolecules in the cell (“molecular crowding”) (264).
11.1 Helix-helix interactions
The final structure of multi-spanning membrane proteins and membrane protein complexes depend critically on interactions between transmembrane helices. Occasionally, these interactions are also important for proper membrane integration of transmembrane helices within multi-spanning membrane proteins (175, 176, 209, 265). The interactions taking place among the helices allow for a higher tolerance for helices containing more polar residues (266).
11.2 van der Waals interactions
The parts of membrane proteins that are embedded in the lipid environment contain mostly non-polar residues. The side chains of these residues interact through weak van der Waals interactions (induced dipole-dipole interactions) (49, 66, 267).
11.3 Salt bridges
When two residues in a protein with opposite charges interact they form a salt bridge. The insertion of charged amino acids in transmembrane helices comes with a large energetic cost. Thus, the number of these amino acids is low, and they often interact with each other through salt-bridges. Salt bridges between transmembrane helices may affect the protein function. An inter- helical salt bridge in the Lac permease of E. coli is one example (268).
11.4 Hydrogen bonds
Polar residues in transmembrane helices form hydrogen bonds to each other, thus contributing to helix-helix interactions (61, 173, 269, 270). Hydrogen bonds occurring between charged or polar residues are quite strong. However, weaker hydrogen bonds can also form by interactions between polar atoms located in the protein backbone, particularly involving small
47 residues such as Ala and Gly that can form weak CĮ –H…O hydrogen bonds (271).
11.5 The GxxxG motif
The GxxxG motif, where three residues separate two glycines in a transmembrane helix is a well characterized common motif important for helix-helix packing. The two glycines in one helix create a cavity that the backbone from the GxxxG motif in the neighboring helix can fit in. This sort of interaction leads to a right-handed crossing angle and is stabilized by van der Waals interactions. However, due to the tight packing of the helices, additional stabilizing forces can be contributed by weak CĮ –H…O or CĮ – H…O=C hydrogen bonds (271, 272). Tandem GxxxG motifs in helices have been found to trigger extensive interactions between helices (224, 273). The GxxxG motif or its other variants (G/A/S)xxxGxxxG and GxxxGxxx(G/S/T) have been identified in more than 10% of all known helix-helix interaction sites in membrane proteins (274). The same motif but with an antiparallel version is proposed to occur in 16 % of the helix packing sites of membrane proteins (275).
11.6 Knobs-into-holes packing
Approximately 30 % of all helix pairs form left-handed anti-parallel packing interactions, where knob-into-holes packing stabilizes the structure (275). The side chains belonging to one helix protrude into cavities on the other helix.
48 12 Translational arrest peptides
Certain short peptide sequences (typically 15-20 residues) have been shown to be able to stall translation during their own synthesis. These are called translational arrest peptides (APs) and have been found in different organisms, mostly bacteria. In the early 1980s APs were shown for the first time to act as regulatory segments involved in antibiotic resistance (276, 277). Translation arrest has been found to be an effect induced by the amino acid rather than the mRNA sequence. By stalling translation on a specific codon, APs control where and for how long the ribosome sits on the mRNA. This can influence different events taking place on the mRNA and thereby affect gene expression (278).
49
mainly negatively charged character, and can interact with the side chains of a nascent polypeptide chain (297, 298).
12.3 Interactions between the ribosomal tunnel and passing nascent chains
As nascent polypeptides pass through the ribosomal tunnel, specific interactions can occur between the amino acids and the ribosomal components lining the tunnel wall. This may affect the conformation of some nascent polypeptides in the way that both allows helix and non-helix formation to take place and also induce conformational rearrangements in the nascent chain up to 45 Å away from the site of interaction in the tunnel (288, 295, 298-301). The ancient ribosome complex may have evolved a cavity within its own structure, in order to manage to protect the synthesized product from a surrounding milieu (302). The cavity may have subsequently evolved into a more sophisticated structure, namely the tunnel, which developed the capability to analyze and respond to nascent polypeptides, by e.g. pausing translation upon synthesizing an arrest peptide sequence (278).
12.4 Nascent-chain induced translational stalling
In vitro studies have shown the entry of aminoacyl-tRNAs into the ribosomal A-site is a rate-limiting step during translation (303). In vivo, elongation rates of polypeptides can vary substantially. This variation can have several reasons, such as codon usage (304) and mRNA secondary structure (305). With the use of the ribosomal profiling technique, information has been collected reflecting the distribution of ribosomes on mRNAs undergoing translation at different time points (285, 306). In particular, such experiments have identified ribosomal pause sites of different duration. The data is in line with experimental results showing frequent pausing events occurring in both bacterial and eukaryotic cells not caused by biased codon usage (286, 307). The side chains of residues situated in either the A- or P-site of the ribosome influence the rate of chain elongation (308, 309). In addition, up to two residues appearing before the stop codon act on the termination process, and with proline present at that site, the termination will acquire the slowest possible rate (310, 311). In eukaryotes, stretches of positively charged residues in a nascent chain have been found to slow down the rate of elongation (312, 313).
52 12.5 Features of arrest peptides
Key residues dispersed along the arrest peptides have been identified as to be essential for the translational pausing event to occur (279). Translational arrest takes place during elongation at the peptidyl transfer step. Thus the peptidyl-tRNA stays in the ribosomal P-site. This is the case for the SecM, MifM and ErmCL-erythromycin arrest peptides (314-316). SecM requires a certain combination of amino acids situated in the P- and A-site in order to induce stalling (314), whereas, the last residue in the ErmCL arrest peptide located in the P site does not have the capability to react with any amino acid positioned in the A-site (317). Translational pausing can also take place during the elongation step, where the peptidyl-tRNA is left in the ribosomal A-site (318). The TnaC-tryptophan and arginine attenuator peptide (AAP) inhibits the termination reaction and only works when there is a stop codon in the ribosomal A-site (319, 320).
12.5.1 The SecM arrest peptide
APs can be classified into inducible and intrinsic APs. An inducible AP pauses translation in response to the binding of a small molecule such as an antibiotic. In contrast, intrinsic APs arrest mRNA translation by default through interactions within the ribosome tunnel (52). The E.coli secretion monitor protein (SecM) AP belongs to the intrinsic AP category and is composed of 17 residues (FSTPVWISQAQGIRAGP) (52). One operon encodes both SecM and the translocation motor protein SecA. SecA is responsible for driving the movement of proteins through the SecYEG translocon machinery in the inner membrane (52). When this process of protein secretion is not functioning properly SecM helps up-regulate the expression levels of the SecA protein (321) (322). SecM contains an N-terminal signal sequence that targets the ribosome-nascent chain complex (RNC) to the SecYEG translocon. Co-translational translocation of SecM , driven by SecA, exerts a force on the nascent chain that prevents ribosome stalling on the AP. Under conditions when the SecA- SecYEG machinery is compromised or overloaded, SecM RNCs become mis-localized to the cytosol where they undergo translational pausing (323). As ribosomes pause on the secM and secA mRNA, the secondary structure of the mRNA gets disrupted. This leads to the exposure of the secA Shine- Dalgarno sequence, thus increasing translation of the SecA open reading frame (322, 324, 325) (Fig. 21).
53
However, as the HR2 exits the ribosome tunnel in order to target the unspliced XBP1 mRNA RNC to the membrane, only 13 codons are present at the ribosome for translation before reaching to the stop codon. At the C terminal part downstream of HR2 the unspliced XBP1 protein follows with a length of 53 amino acids (283). With the length equivalent to 40 amino acids in the ribosomal tunnel, this leads to unspliced XBP1 protein being completely produced and detached from the complex right after HR2 emerges from the ribosomal tunnel (327). In order to prevent this release to occur, the 21 residues long XBP1 AP (YQPPFLCQWGRHQPSWKPLMN) inside the ribosome stalls the translational elongation of the unspliced XBP1 protein at the point when HR2 is exposed from the tunnel. The arrested state allows enough time for the co-translational targeting mechanism to take place while the RNC is still fully assembled (283).
12.5.3 Release of arrest-peptide mediated translational stalling
The interactions between arrest peptides and the inner wall of the ribosome exit tunnel can be broken by interactions between the nascent chain and factors located outside the ribosome. Thus, co-translational processes can affect translational stalling. Two models initially explained the release of the SecM arrest peptide from the ribosome tunnel. The first suggested that the presence of an external pulling force on the nascent chain releases the arrested state. The second postulated a signal transduction model where an externally induced conformational change in the ribosome would release the stall (325, 328). It is now clear that the first model is the correct one, at least for SecM. Even when transplanted into other proteins, the SecM and XBP1 arrest peptides stall translation, unless there is an external pulling force on the nascent chain (329, 330). In paper III (330), we show that a hydrophobic transmembrane segment generates a sufficiently strong pulling force during its co-translational membrane insertion to overcome the interactions between both the bacterial SecM and the eukaryotic XBP1 arrest peptides and the inner wall of the ribosomal tunnel, but only if it is located between 30 and 40 residues upstream of the arrest peptide. Thus, arrest peptides can be used as force sensors to follow the co-translational membrane insertion of transmembrane segments.
12.5.4 Other amino acid clusters can arrest translation
The negative surface charge inside the tunnel can cause retention of positively charged nascent chain segments in the ribosome. In particular, the poly(A)tail in aberrant mRNAs that lack a stop codon gets translated into a cluster of lysine residues, which results in a pausing event during elongation
55 and triggers nonstop and/or no-go mRNA degradation and proteolysis via ubiquitination of the arrested protein fragment (313, 331, 332). 8-12 arginines or lysines have been found to be sufficient to cause translational mRNA-stalling. As few as three translated arginine residues near each other can induce detectable translational arrest as a consequence of electrostatic interactions with the negatively charged ribosome tunnel wall (312).
12.5.5 Laboratory-engineered arrest peptides
New ribosomal arrest peptide sequences have been engineered, with the purpose of both expanding the toolbox of useful arrest peptides and in an effort to understand the diverse interactions taking place between arrest peptides and the ribosome exit tunnel (333, 334).
56
14 Summary of publications
14.1 Paper I - Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences
0RVWRIWKHĮ-helical shaped proteins found in the membrane of prokaryotic and eukaryotic cells enter the membrane through the lateral gate of the main SecYEG/Sec61 translocation machinery. The gate is thought to open up to the core of the lipid bilayer and allow polypeptide chains to partition into the membrane. The amino-acid sequence of protein segments dictates the efficiency of the membrane insertion. Studies analyzing the influence of individual residues as well as overall hydrophobicity, flanking charges and length of the transmembrane segments on protein sequence that are aimed for membrane insertion have been carried out in both in vitro and in vivo (353, 354). A previous study by Demirci et al. demonstrated that a cluster of Leu residues in a poly-Ala background increased the membrane insertion efficiency in vivo more than when the same number of Leu residues were dispersed along the segment (355). Thus, a transmembrane segments hydrophobicity can be affected by the order of residues along a local sequence, its tendancy for forming secondary structures and its water and membrane milieus. However, still the process for TM segment identification and insertion is still not well understood. In this paper we addressed the issue of the importance of hydrophobic patterning both for peptide partitioning into membrane-mimetic environments (SDS, reverse-phase HPLC, reconstituted liposomes) and for translocon-mediated membrane insertion. We created a library of a set of peptides composed of different sequences but with identical amino acid composition. The average hydrophobicity of the peptides was chosen to be appropriate for membrane insertion, according to the Liu-Deber hydropathy scale (356). We found that peptides containing blocks of Leu residues, e.g. LLLLLLLLLWSSSSSS, partitioned more efficiently into membrane-like environments than peptides containing discontinuous regions of Leu residues, e.g. SLSLLSLSSWSLLSLSLLS. Thus, hydrophobic patterning contributes in a similar way to membrane insertion in vitro and in vivo.
63
14.3 Paper III - A Biphasic Pulling Force Acts on Transmembrane Helices During Translocon- Mediated Membrane Integration
The majority of membrane proteins in both prokaryotic and eukaryotic cells insert into the lipid bilayer in a co-translational manner through the SecYEG/Sec61 translocon (25). The energetics during membrane protein insertion is quite well characterized (163, 353, 358), but the dynamics of insertion is yet not fully understood. In this paper, we aimed at identifying local forces generated on a hydrophobic transmembrane segment in a nascent polypeptide chain during the insertion process, both in vivo in E. coli and in mammalian rough microsomes. We recorded forces acting on the nascent chain during co-translational integration by using arrest peptides as force sensors (279). The prokaryotic arrest peptide used in the in vivo assay was from the SecM protein (359). The mammalian arrest peptide used in the in vitro assay was XBP1 (360). We observed strong pulling forces exerted onto nascent chains including a transmembrane helix with a hydrophobicity that would be suitable for membrane insertion. The pulling forces overcame the translational stalling capacity of the arrest peptides in the ribosomal tunnel such that translation continued beyond the arrest peptide. The pulling force showed two maxima when the C-terminal end of the transmembrane helix was approximately 30 and 40 residues away from the PTC. We hypothesized that the N-terminus of the transmembrane helix engaged in a first interaction with the translocon or lipid-translocon interface region at the shorter distance of 30 residues, whereas, the second, stronger maxima at the distance of 40 residues represents the step when the transmembrane helix partitions into the membrane; interestingly, the first maximum is not seen in the force profile recorded in the eukaryotic rough microsome system. By varying the hydrophobicity of the transmembrane segment, we found that overall hydrophobicity is the main determinant of the pulling force exerted on the nascent chain during co-translational membrane integration.
14.4 Paper IV – Mutational Analysis of the Human XBP1 Translational Arrest Peptide and Construction of Arrest-Enhanced Variants
During protein synthesis, the nascent chain exits the ribosome in a 100 Å long tunnel in the large ribosomal subunit (361). However, certain protein sequences – so called translation arrest peptides – have been found to
65 interact with the exit tunnel and the PTC, causing translational pauses of varying durations (295, 346, 359, 362, 363). XBP1 is the most studied mammalian arrest peptide. It is localized in the XBP1 protein, which regulates the unfolded protein response (360). In paper III, the XBP1 arrest peptide was used as a force sensor to record pulling forces during membrane protein integration into rough microsomes. However, the XBP1 arrest peptide is quite long and has only a rather weak arrest potency. These considerations raised the question whether we could construct arrest- enhanced XBP1 arrest peptide variants by saturation mutagenesis. Our starting construct was from paper III, containing a linker distance of 43 residues downstream of the PTC. The results showed that many amino-acid substitutions generated stronger XBP1 arrest peptide variants. One such variant was used to record force profiles for membrane protein insertion into rough microsomes, and to follow the co-translational folding of the ADR1a zinc-finger domain that had previously been analyzed in the E. coli-derived PURE in vitro translation system. Both in E. coli and in the mammalian system, ADR1a was found to fold deep inside the ribosome tunnel (364).
66 15 Final thoughts
In this thesis, we have added to our knowledge about how the patterning of hydrophobic and hydrophilic residues affect the interaction of protein segments with biological membranes and membrane mimetic systems, and have also studied the forces that act on nascent membrane proteins during co-translational membrane integration. In the future, it would be interesting to ask questions such as: Why do transmembrane segments in membrane proteins contain both the hydrophobic Leu and IIe? What differences or similarities in membrane integration would the presence of these two residues result in? When comparing prokaryotic and eukaryotic force profiles, a difference is clearly seen in the early stage of membrane protein integration into the lipid bilayer. It would be interesting to find out more about the reasons for the lack of an early pulling force in the eukaryotic system. Does the transmembrane helix initially interact more with the translocon or the lipids surrounding the translocon? How would the pulling force profiles turn out if amino-acid substitutions would be made inside the translocon? Would this possibly identify residues in the translocon that influence the insertion process? With the newly constructed library of XBP1 arrest peptides, ranging from weaker to stronger translational stalling strength, it would be interesting to study their interactions with the ribosome exit tunnel by cryo-EM. Our collection of high potency arrest peptides, now make it possible to study a wide range of co-translational events such as different protein-protein/ or other cell component interactions in mammalian systems. In the future, it would also be interesting to identify other mammalian arrest peptides and carry out a comparative analysis of residue sequence and structure to elucidate similarities and differences between different arrest peptides.
67 16 Acknowledgements
While writing this last section of my thesis I am realising that my Ph.D. student journey is soon coming to its end. This leaves my heart filled with both relief and grief feelings. During the years of working at the department of biochemistry and biophysics at Stockholm University I have had the pleasure of meeting many great and inspiring people. Thank you all for creating a fun and scientifically outstanding environment. I would like to give a special thank you to people who contributed in the making of this thesis. First and foremost I thank you Gunnar for being such an excellent, thoughtful and good-hearted scientist and supervisor. It has been an honor to work in your research group. Thank you for all your support and happy memories throughout these years! Many thanks to you IngMarie for all your scientific help and willingness to share your huge knowledge in experimental works. I am grateful that I got to know you. Thank you Stefan Nordlund, Pia Ädelroth, Daniel Daley, Martin Högbom, Joe DePierre, Peter Brzezinski and Martin Ott for your help throughout all evaluations during the Ph.D. studies. Thank you Agneta Norén, Pia Harryson and Inger Carlberg for all guidance during the summer research schools and student course teachings. Thank you all co-authors for sharing your knowledge. It has been a pleasure working with you. Thanks to all the present and former GvH and IMN group members and the GvH community for contributing to a fun and inspiring working place. I would like to give a special thanks to: Pilar for being such a sweet friend and all your help and support both inside and outside of the lab; Salomé for all laughs and fun discussions. I miss our lunches at Lantis! Patricia (Flipper) for all laughs both inside and outside of the lab. I have missed having my bench-mate next to me for a while now; Anna for your kindness and random chats and laughs; Rickard, Bill, Florian, Nurzian and Johannes for all the laughs, Lantis lunches, discussions, friendly and great working environment; Carmen for your kindness, scientific knowledge and relaxed personality; Annika, Ola, Karin, Felix, Luigi, Grant, José, Nir and Renuka for a great working environment; Hena for always being so kind, hardworking and helpful; Patrik, for all the laughs and discussions; all help, talks and a lot of laughter from the next door people, Catarina, Erika, Isolde, Johan, Diogo, Åsa, Theresa and the sweet Pedro-Beata couple;
68 my former master student and now Ph.D. student Anastasia for your great help in our paper, all laughs and talks about life; Thank you for always being so helpful with a smile on your face; my other students Flavia, Karoline, Maikel, Amanda for your happiness, kindness and hard lab work; some of the group leaders of our floor Elzbieta Glaser, Jan-Willem de Gier, Robert Daniels, IngMarie Nilsson and Daniel Daley for your scientific- friendly advices, help with lab equipment and materials and your passion for great science. Many thanks to Ann, Maria (for sooo much laughter and great talks), Malin, Charlotta, Alexander, Matthew, Ann-Britt, Håkan, and Peter for keeping the department running and for being very helpful with all questions from the rest of the DBB-staff! To my dear friends outside of work: Carolina, for our long-lasting friendship of more than 3 decades. Thank you for always making me laugh through life even when you are miles and miles away from home; Veronica, for all wonderful dinners, fun nights out and laughs through the years after the summer research school; Sophie, for your kind and joyful friendship after the birth of our sweet girls. I look forward to many more laughs with you and your cute family in the future; Rasmus, for all fun dinners, parties, laughs and discussions. My lovely parents-in-law, Ulla and Göran, thank you for all your support in my Ph.D. studies and curiosity in what I am working with in the lab. Mom and dad, without your love and support that you have given me during my life, I would not be the person I am today. I love you! My dear husband, Christian, expressing my gratitude for all your love and support for me during all the years of studying is like kissing you – there can never be enough of it. Ich liebe dich! Isabelle och Ellinor, my sweet bundles of joy. I love your every cell!
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